Fe3O4-Polyaniline Nanocomposite for Non-enzymatic Electrochemical Detection of 2,4-Dichlorophenoxyacetic Acid.

This study proposes the development of an electrochemical sensor based on fabrication of a glassy carbon electrode (GCE) with Fe3O4-polyaniline (Fe3O4-PANI) nanocomposite, which was further used for enzyme-less detection of 2,4-dichlorophenoxyacetic acid (2,4-D) in aqueous medium. Spectroscopic studies, microstructural studies, and elemental analysis established the formation of Fe3O4 nanoparticles with polyaniline coating. The fabricated Fe3O4-PANI-GCE was characterized by electrochemical techniques like cyclic voltammetry and electrochemical impedance spectroscopy. The electrochemical response of 2,4-D on Fe3O4-PANI-GCE was evaluated by performing cyclic voltammetry and amperometry experiments. The synergistic effect of the composite causes the superior electrochemical behavior of Fe3O4-PANI-GCE toward the detection of 2,4-D. Amperometric measurements exhibited a linear concentration range from 1.35 to 2.7 μM. The sensitivity and detection limit were evaluated from the amperometric responses, which were found to be 4.62 × 10-7 μA μM-1 cm-2 and 0.21 μM, respectively. The electrochemical sensing response could be attributed to adsorption of 2,4-D onto the Fe3O4-PANI-modified GCE (Fe3O4-PANI-GCE) surface. Fe3O4-PANI-GCE is found to be a simple, low-cost, and biocompatible non-enzymatic sensor for detection of 2,4-D in aqueous medium at ambient temperature.

Introduction

The increase in food demand due to population outgrowth has seen a significant rise in the use of pesticides and herbicides in the agriculture sector. However, their extensive use has a serious negative impact on living organisms as well as the environment.[1] 2,4-Dichlorophenoxyacetic acid (2,4-D) is an auxin-like herbicide used conventionally for controlling growth of broad-leaf weeds in agricultural fields.[2−4] 2,4-D mimics the action of auxin (a plant growth hormone) and induces uncontrolled growth and death of weeds.[2] It is also found as an active ingredient in many pesticides, and improper usage may prove fatal to human health even at trace amounts as it leads to excess release of reactive oxygen species.[5] Overuse of this herbicide has also led to its persistence in water sources due to its high solubility and nonbiodegradable nature.[1,6] Even a concentration less than 3 mg L–1 in drinking water imparts an unpleasant taste and odor.[6] The herbicide has been identified as a human carcinogen and a toxic pollutant by the Environmental Protection Agency (EPA) and International Agency for Research on Cancer (IARC).[7,8] Keeping view of the hazardous nature of this organic contaminant, it is important to develop efficient, reliable, and cost-effective techniques for convenient and accurate detection of herbicides in various water sources especially in drinking water. Till date, many techniques have been employed to determine the presence of herbicides like 2,4-D in water and environmental samples. These methods of detection involve liquid chromatography, gas chromatography, immunoassay techniques, photo-electrochemical sensing, capillary electrophoresis, and potentiometric as well as electrochemical methods.[6] Although these methods are highly sensitive and provide accurate results, they come with certain limitations like high cost and requirement of pretreatment and expertise to obtain accurate results.[9] Electrochemical methods have gained more popularity due to their simplicity, low cost, sensitivity, and rapid response time. This method also has an added advantage of miniaturization and on-site monitoring of samples. Electrochemical methods are based on a reaction on the electrode surface due to transfer of electrons.[6,10,11]

Conducting polymers have potential sensing applications owing to their flexibility, high sensitivity, excellent redox properties, low cost, and ability to perform at room temperature.[2,12−14] Polyaniline (PANI) with its versatile properties has emerged as a potential conducting polymer in the field of electrochemistry and sensing.[2,12,15,16] PANI-based composites have a great deal to offer as sensing materials because of their high surface area, π-conjugated structure, excellent redox properties, and presence of amine groups.[17] However, they have certain limitations due to their poor solubility in common organic solvents, instability at higher temperature, poor electrochemical stability, reproducibility, and selectivity.[12,17−19] On the other hand, transition metal oxide nanoparticles owing to their electrocatalytic activities and strong adsorption abilities have received tremendous attention in electrochemical and bio-sensing applications.[20−22] Among various metal oxides, iron oxide (Fe3O4) nanoparticles are gaining more attention in bio-sensing applications due to their favorable band gap, biocompatibility, nontoxicity, thermal stability, interesting optical and magnetic properties, and high natural abundance.[21,23,24] Fe3O4 nanoparticles have novel prospects in the field of electrochemical sensing because of their high electrical conductivity even at room temperature due to electron exchange between Fe2+ and Fe3+ ions.[25,26] However, metal oxides require high temperature for sensing performance.[23] To overcome these limitations, development of organic–inorganic hybrid nanocomposites of metal oxides and conducting polymers may be promising for commercial development of cost-effective electrochemical sensors.[16]

Currently, the detection of 2,4-D is based on an enzyme immobilization technique.[4,27−29] Despite its popularity, an enzyme-based sensing technique comes with certain operational limitations such as high cost, thermal and chemical instability, loss of enzyme bioactivity, short lifetime, and low reproducibility.[9,10,20,30] In this work, we fabricated a glassy carbon electrode modified with iron oxide-polyaniline nanocomposite (Fe3O4-PANI-GCE) for non-enzymatic detection of 2,4-D in aqueous medium. Here, the conductive Fe3O4-PANI nanocomposite acts both as the recognition element and sensor material for electrochemical detection of 2,4-D without the use of electroactive labels. Cyclic voltammograms and amperometric current response were recorded as results of adsorption of 2,4-D molecules on the nanocomposite surface. It was found that Fe3O4-PANI-GCE exhibited an enhanced electrochemical response toward 2,4-D compared to Fe3O4-GCE and PANI-GCE. Detection of 2,4-D was evaluated from the current response of amperometry experiments. Fe3O4-PANI-GCE showed a wide linear range of detection with a low detection limit for 2,4-D with good sensing ability due to its adsorptive capability.

Results and Discussion

Characterization of Fe3O4-PANI

The synthesized materials were characterized by Fourier transform infrared (FTIR) spectroscopy, as shown in Figure a, for identification of functional groups present. The band at 582 cm–1 is the main characteristic band of the Fe–O stretching vibrations in Fe3O4 nanoparticles, indicating the formation of metal oxide.[24,31,32] Fe3O4-PANI spectrum also shows bands at 555 and 634 cm–1, indicating the presence of Fe–O bonds in the composite. Peaks at 839, 1141, 1384, and 1579 cm–1 are due to the presence of PANI in the composite.[15,32] The peak at 1579 cm–1 is due to the C=C stretching vibration of the quinoid rings.[30,33] The 1384 cm–1 peak can be attributed to the C–N stretching vibration of acid-doped PANI. The 1141 cm–1 band is related to the C–N stretching of a secondary aromatic amine.[34] The peak at 839 cm–1 was assigned to the out-of-plane C–H deformation in the 1,4-disubstituted benzene ring.[32,35,36]

Figure 1

(a) FTIR spectra, (b) PXRD pattern of Fe3O4 and Fe3O4-PANI, (c and d) SEM images of Fe3O4 and Fe3O4-PANI, respectively, and (e) FETEM image of Fe3O4-PANI nanocomposite.

Figure b presents the powder X-ray diffraction (PXRD) pattern of Fe3O4 and Fe3O4-PANI. The XRD peaks observed in Fe3O4 nanoparticles are consistent with the magnetite phase of Fe3O4 as per JCPDS No. 96-900-2318 (shown in Figure SI-1). The characteristic peaks at 30.16, 35.52, 43.18, 53.6, 57.14, 62.72, and 74.24° correspond to the (220), (311), (400), (422), (511), (440), and (533) planes of Fe3O4.[22,26,37−39] The sharp intense peaks indicate the high crystallinity of the iron oxide phase.[40] Peak broadening is observed, which is consistent with ultrafine nanocrystalline particle formation.[38] The diffraction peaks can be indexed to the cubic inverse spinel structure, which belongs to the Fd-3m space group.[41,42] The low-intensity peaks of PANI are not observed in the Fe3O4-PANI diffraction pattern as they remain diffused as broad peaks in the presence of the high-intensity iron oxide peaks. It is observed that there is no structural change in Fe3O4 during formation of composite.

Figure c,d illustrates the scanning electron microscopy (SEM) images of Fe3O4 and Fe3O4-PANI composite, respectively. Figure c depicts the formation of iron oxide nanoparticles with a smooth surface. The micrograph of Fe3O4-PANI indicates the formation of a uniform thin layer of PANI on the surface of Fe3O4 nanoparticles. Surface functionalization of the nanoparticles with PANI resulted in globular aggregates with a relatively rougher surface, as seen in the SEM micrograph of Fe3O4-PANI. Formation of PANI coating over Fe3O4 could be linked to electrostatic interaction between PANI and surface charge on iron oxide nanoparticles.[15,38] Thus, the SEM results indicated the successful surface functionalization of the Fe3O4 nanoparticles with PANI. Figure e shows the TEM image of Fe3O4 nanoparticles coated with PANI. The dark regions in the micrograph are due to the presence of magnetite Fe3O4 nanoparticles.

The EDXS patterns of Fe3O4 nanoparticles and Fe3O4-PANI nanocomposite are shown in Figure a,b, respectively, with the inset showing their respective elemental composition tables. EDXS analysis of Fe3O4 shows the presence of 63% of Fe and 27% of O, while Fe3O4-PANI nanocomposite composed of Fe (53%), O (20%), C (11%), N (7%), and Cl (1%) confirms the formation of PANI coating on Fe3O4 nanoparticles. The elemental mappings of Fe3O4 and Fe3O4-PANI composite are shown in Figure c,d, respectively. The presence of the elements like C, N, and Cl in the nanocomposite confirms the formation of PANI coating on the Fe3O4 surface. Thus, from SEM, EDXS, and elemental mapping analysis, the formation of PANI coating on Fe3O4 nanoparticles can be confirmed. The presence of K in Fe3O4 could be traced to the use of KOH in the synthesis process.

Figure 2

EDXS of (a) Fe3O4 nanoparticles and (b) Fe3O4-PANI. The inset shows elemental analysis tables. Elemental mappings of (c) Fe3O4 nanoparticles and (d) Fe3O4-PANI nanocomposite.

Electrochemical Characterization of Fe3O4-PANI-GCE

The electrochemical behavior of the modified GCE was evaluated both in the absence and presence of 2,4-D from the cyclic voltammograms (CV) recorded vs Ag/AgCl as a reference electrode within a potential range of −0.4 V to 0.6 V at a scan rate of 60 mVs–1. In Figure a, the CVs of the bare GCE both in the absence and presence of 0.9 μM 2,4-D do not show any characteristic peak. On the other hand, CVs of Fe3O4-PANI-GCE both in the absence and presence of 0.9 μM 2,4-D showed redox peaks. Fe3O4-PANI-GCE without and with 2,4-D showed peak to peak separation potentials (ΔEp) of 0.163 and 0.165 V, respectively, which confirmed its electroactive behavior and quasi-reversible electrode reaction.[43,44] The redox peaks observed in Fe3O4-PANI-GCE is due to faradaic behavior of polyaniline.[45] PANI in association with Fe3O4 nanoparticles shows sharp redox peaks, indicating enhanced electroactivity and conductivity.[46] Low ΔEp values indicate the occurrence of reversible charge transfer processes at the electrode surface.[47] For Fe3O4-PANI-GCE in the absence of 2,4-D, the oxidation or anodic peak potential (Epa) is observed at 0.215 V and the reduction or cathodic peak potential (Epc) at 0.052 V. However, addition of 2,4-D resulted in shifts of Epa and Epc to 0.255 and 0.1 V, respectively. These shifts in peak potentials can be attributed to the diffusion and kinetic limitations of the analyte at the electrode–electrolyte interface due to the adsorption of 2,4-D on the nanocomposite surface.[11,24]

Figure 3

(a) CVs of the bare GCE and Fe3O4-PANI-GCE in the absence and presence of 2,4-D and (b) Nyquist plots for bare GCE and Fe3O4-PANI-GCE [the inset shows the magnified Nyquist plots].

The enhanced redox peak currents observed in the CV response of Fe3O4-PANI-GCE as compared to bare GCE can be attributed to the electrochemical property of Fe3O4-PANI nanocomposite, which can be further verified from electrochemical impedance analysis. Electrochemical impedance spectroscopy (EIS) helps in analyzing interfacial properties of electrode materials and provides an insight regarding the charge transfer behavior of modified electrode surfaces.[47]Figure b shows the Nyquist plots for bare GCE and Fe3O4-PANI-GCE in 0.1 M PBS with a frequency range of 0.01–105 Hz at a constant potential of 100 mV. The electron charge transfer resistance (Rct) values as measured from Nyquist plots [shown in Figure b] were found to be 68.9 and 57.5 Ω for bare GCE and Fe3O4-PANI-GCE, respectively. It has also been observed that the semicircle portion of the Nyquist plot for Fe3O4-PANI-GCE has reduced and has become almost a straight line. The low resistance value and reduction of the semicircle portion for Fe3O4-PANI-GCE are indications of an accelerated electron transfer process leading to good electrical conductivity.[17] The increase in electron transfer capability and the redox reaction exhibited by the modified GCE can be attributed to the synergistic effect between Fe3O4 and PANI in the nanocomposite where PANI is acting as a redox mediator.[11,25,47−49] The lower Rct value for Fe3O4-PANI-GCE is a consequence of excellent conductivity of Fe3O4 and PANI in the nanocomposite.[15] In addition, the presence of PANI in the nanocomposite prevented aggregation of nanoparticles, resulting in an augmented electrochemical surface area providing a good number of active sites that facilitates the interfacial charge transfer capability.[36,50] The Nyquist plots presented in the Supporting Information [Figure SI-2] also support the lower charge transfer resistance of Fe3O4-PANI-GCE than Fe3O4-GCE and PANI-GCE.

Electrochemical Response of 2,4-D at the Fe3O4-PANI-GCE Surface

CVs of Fe3O4-PANI-GCE recorded at different scan rates in the absence of 2,4-D [Figure a] showed an increase in oxidation and reduction peak currents with increasing scan rates. The occurrence of redox peaks demonstrates the electroactivity of the modified GCE. With faster scan rates, higher current densities are observed, which could be due to a decrease in the size of the diffusion layer.[51] Shifting of reduction peaks toward lower potential and oxidation peaks toward higher potential [Figure a] with increasing scan rates demonstrates good reversibility of Fe3O4-PANI due to weak polarization.[50]Figure b shows the linear plots of Ipa and Ipcvs ν1/2 (square root of scan rate). Both the oxidation and reduction currents increase linearly with increasing square root of scan rate, which indicates diffusion-controlled electron transfer kinetics at the Fe3O4-PANI-GCE surface.[47]

Figure 4

(a) CVs of Fe3O4-PANI-GCE with different scan rates (20–80 mVs–1). (b) Calibration plot for the linear dependence of oxidation and reduction peak currents vs square root of scan rate. (c) CVs of Fe3O4-PANI-GCE with successive 2,4-D addition (0.22–4.5 μM) in 0.1 M PBS (pH 7). (d) Calibration plot for the reduction current variation with 2,4-D concentration. (e) Nyquist plots for Fe3O4-PANI-GCE before and after addition of 0.9, 1.8, and 2.7 μM 2,4-D [inset showing magnified Nyquist plots].

The response of the modified GCE (Fe3O4-PANI-GCE) toward the herbicide 2,4-D as presented in Figure c shows an electrochemical redox process with oxidation and reduction peaks at 0.255 and 0.1 V, respectively. CVs were recorded at a scan rate of 60 mVs–1 within a potential range of −0.4 to 0.6 V with successive addition of the analyte (2,4-D) in a concentration range of 0.22–4.5 μM. A decrease in the current density was observed with an increase in analyte concentration. With successive addition of 2,4-D, a relative decrease in current density in the CVs was seen. The adsorbed 2,4-D species on the surface of the electrode forms a barrier reducing the conductivity of the PANI surface, which contributes to the decreasing rate of electron transfer at the electrode–electrolyte interface, resulting in decreased current density. The CV response of 2,4-D was also tested on PANI-modified GCE (PANI-GCE) [shown in Figure SI-3] and Fe3O4-modified GCE (Fe3O4-GCE) [shown in Figure SI-4], which showed no significant changes in the redox peak currents. This exhibited that Fe3O4-PANI-GCE showed an enhanced electrochemical response toward 2,4-D due to the synergistic effect of both the components, i.e., Fe3O4 and PANI, compared to pristine Fe3O4 and pristine PANI.

A calibration curve of reduction peak current vs 2,4-D concentration was plotted [shown in Figure d] from the corresponding CV responses of Fe3O4-PANI-GCE to 2,4-D. The plot, as shown in Figure d, indicates a good linear behavior (R = 0.98) of the reduction current vs 2,4-D concentrations within the range 0.22–4.5 μM.

Electrochemical impedance spectroscopy of Fe3O4-PANI-GCE was also conducted before adding the analyte and after 0.9, 1.8, and 2.7 μM 2,4-D addition at a frequency range 0.01–105 Hz. Figure e presents the Nyquist plots showing an increase in Rct values with successive 2,4-D addition. This indicates the increase in charge transfer resistance of the sensor material on exposure to the analyte, which tends to adsorb on the electrode surface, giving rise to a higher Rct value. This also indicates a direct relationship between the electrochemical impedance signal and the amount of analyte adsorbed on the electrode surface.[24]

Mechanism

A plausible non-enzymatic sensing mechanism for 2,4-D with Fe3O4-PANI-GCE can be ascribed to adsorption of the 2,4-D anionic species at pH 7 on the PANI surface of the nanocomposite followed by proton exchange (i.e., protonation and deprotonation) between the N–H group of PANI and the anionic species of 2,4-D, which exist at pH 7.[12,34] This analyte interaction with the electrode surface resulted in increased resistance (Rct) of Fe3O4-PANI-GCE, which can also be deduced from the Nyquist plots in Figure e.

The decrease in redox current could be due to (i) π–π electron coupling between π- electrons of 2,4-D and PANI facilitating electron transfer; (ii) the presence of amine groups on the PANI chain behaving as electrostatic anchoring sites for 2,4-D; and (iii) formation of hydrogen bonds between 2,4-D and PANI.[17,19] These cause adsorption of 2,4-D on the electrode surface, forming an insulation layer that results in an increase in charge transfer resistance for the redox reactions.

Non-enzymatic Amperometric Detection of 2,4-D

Amperometric i–t measurements were performed with different 2,4-D concentrations (0.45–4.05 μM) at a fixed potential of 0.1 V in 0.1 M PBS (pH = 7). Figure a shows the amperometric response of Fe3O4-PANI-GCE to different 2,4-D concentrations, and Figure b shows the corresponding analytical linear graph (current vs concentration) for amperometric response. A linear concentration range was observed from 1.35 to 2.7 μM with a linear equation as follows: I = 3.24 × 10–8x- 8.53 × 10–8. The reduction current is directly proportional to the concentration of the analyte, which increases linearly (R = 0.97) with an increase in concentration and gets saturated at higher concentration. This observation is in agreement with the CV response of 2,4-D with different analyte concentrations toward Fe3O4-PANI-GCE. The limit of detection (LOD) can be obtained from the formula presented in eq (6,36,47)where σ is the standard deviation of the data points and s is the slope of the linear plot (current vs concentration). The LOD was calculated to be 0.21 μM. A comparison table [Table SI-1] with LOD values is provided in the Supporting Information, which highlights some of the reported works based on different detection techniques for 2,4-D. The present work reports a quite good LOD value as compared to many other reported electrochemical sensors that operate without the use of electroactive labels.

Figure 5

(a) Amperometric response of 2,4-D (0.45–4.05 μM) with Fe3O4-PANI-GCE at 0.1 V in 0.1 M PBS and (b) analytical linear graph representing current vs concentration obtained from amperometric response.

The sensitivity of Fe3O4-PANI-GCE can be obtained from the slope of the linear plot of amperometric response and the area of the electrode.[52]Equation is used for calculating the sensitivity[53]where the slope is obtained from the linear amperometric calibration response plot as shown in Figure b and the area of the electrode was calculated to be 0.07 cm2 (diameter of 3 mm). The sensitivity of the sensor was found to be 4.62 × 10–7 μA μM–1 cm–2.

Conclusions

This work illustrated the fabrication of GCE with Fe3O4-PANI nanocomposite. This modified GCE was effective for non-enzymatic electrochemical detection of the herbicide 2,4-D at neutral pH. The electrochemical techniques of cyclic voltammetry and amperometry were employed for electrochemical detection of 2,4-D. Calibration of amperometric current response showed a linear concentration range from 1.35 to 2.7 μM of 2,4-D. A low limit of detection of 0.2 μM and sensitivity of 4.62 × 10–7 μA μM–1 cm–2 were obtained from the analysis of the amperometric responses. Here, the simple adsorption of the 2,4-D molecules on the surface of Fe3O4-PANI nanocomposite is responsible for detection of 2,4-D. Further development of advanced electrochemical sensing devices may be expected based on this mechanism. This Fe3O4-PANI-modified GCE offers to be a simple and low-cost non-enzymatic sensor for 2,4-D.

Experimental Section

Materials and Methods

Aniline and 2,4-dichlorophenoxyacetic acid were obtained from Sigma Aldrich. Chitosan was bought from National Chemicals. Ammonium peroxodisulfate (≥98%), hydrochloric acid (35–38%), iron(III) nitrate nonahydrate (≥98%), acetic acid, methanol (99.8%), potassium hydroxide (≥84%), sodium hydroxide (≥97%), sodium phosphate dibasic, and sodium phosphate monobasic hydrate were purchased from Merck, India. Ethylene glycol (98%) was bought from Thermo Fischer Scientific. Sodium phosphate dibasic (Na2HPO4) and sodium phosphate monobasic hydrate (NaH2PO4·H2O) were used for preparing 0.1 M phosphate buffer solution (PBS) of pH 7. All the chemicals were used as received except aniline, which was double distilled before use. All the experiments were carried out using distilled water.

FTIR spectra were recorded in the range 4000–500 cm–1 with a Shimadzu, IR Affinity-1 spectrophotometer. Potassium bromide (KBr) was used for preparing the sample pellets for FTIR analysis. Phase identification of the magnetic materials was done from PXRD patterns recorded with a powder X-ray diffractometer (Bruker D205505) using a monochromatic X-ray beam with CuKα (λ = 1.54 Å) radiation scanned at a range of 2θ = 5–80°. Morphologies of Fe3O4 nanoparticles and Fe3O4-PANI nanocomposites were studied with the help of SEM (ZEISS SIGMA 300) and field emission transmission electron microscopy (FETEM, JEOL, JEM 2100F).

Electrochemical measurements (cyclic voltammetry, electrochemical impedance spectroscopy, and amperometric i–t measurements) were conducted with a CHI608E electrochemical workstation in 0.1 M PBS as an electrolyte at room temperature. A setup of three-electrode electrochemical cell was used with a glassy carbon electrode (GCE, 3 mm diameter, 0.07 cm2 surface area) where the active material was deposited as the working electrode, platinum foil as the auxiliary or counter electrode, and Ag/AgCl (KCl, 3 M) as the reference electrode.

Synthesis of Fe3O4-PANI Magnetic Nanocomposite

Fe3O4 nanoparticles were synthesized via a solvothermal process.[54] Surface functionalization of these synthesized nanoparticles with PANI was achieved by chemical oxidative polymerization of aniline in acidic medium (1 M HCl) in the presence of ammonium peroxodisulfate. The synthesis scheme for Fe3O4 and Fe3O4-PANI is discussed in the Supporting Information SI 1.

Fabrication of the GCE with Fe3O4-PANI

Initially, the GCE was cleaned by polishing it with an aqueous slurry of alumina on a polishing pad. The electrode was then ultrasonicated in distilled water for 5 min and left to air-dry. A thin layer of Fe3O4-PANI coating was deposited on the surface of the GCE by a drop-cast method. Prior to drop-casting, 2 mg of Fe3O4-PANI was added to 400 μL of 0.5% chitosan solution (prepared in 1% (v/v) acetic acid solution) and ultrasonicated for 30 min to obtain a homogeneous dispersion. A total of 2 μL of this dispersed material was drop-casted on the surface of the GCE and allowed to air-dry at room temperature. The amount of Fe3O4-PANI deposited on the GCE surface was estimated to be 0.01 mg. This modified GCE (Fe3O4-PANI-GCE) was further used for conducting the electrochemical experiments. Figure shows a schematic representation of GCE modification by drop-casting of Fe3O4-PANI and the setup for electrochemical detection of 2,4-D.

Figure 6

Schematic representation of the glassy carbon electrode (GCE) modification by drop-casting Fe3O4-PANI and the setup for electrochemical detection of 2,4-D.

Electrochemical Experiments

For conducting the electrochemical experiments, a phosphate buffer solution of 0.1 M concentration was prepared using sodium phosphate dibasic (Na2HPO4) and sodium phosphate monobasic hydrate (NaH2PO4·H2O) and the pH of the solution was adjusted at 7. A 1000 mg/L stock solution of 2,4-D was prepared in methanol. The required 2,4-D analyte of 100 mg/L was prepared by further dilution of the stock solution with distilled water.

Electroanalytical techniques used in this work were cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and amperometry. CV was used for characterization of the modified GCE with respect to the unmodified GCE and also to study the electrochemical response of Fe3O4-PANI-GCE toward 2,4-D. EIS of bare GCE and Fe3O4-PANI-GCE both in the absence and presence of 2,4-D was studied to evaluate their charge transfer resistance. Amperometric measurements were done for optimization and quantification of 2,4-D, which were performed at a specific potential (determined from the CV experiments) in 0.1 M PBS.