Electrochemical Detection of Diclofenac Using a Screen-Printed Electrode Modified with Graphene Oxide and Phenanthroline

Abstract

In recent years, interest in screen-printed electrodes (SPEs) has grown due to their wide range of applications. Diclofenac (DCF), a widely used non-steroidal anti-inflammatory drug, is a subject of interest in pharmaceutical research as well as environmental research, primarily due to its environmental contamination and therapeutic applications. This study describes the development and characterization of an innovative screen-printed sensor based on graphene oxide (GO) and phenanthroline (PHEN) for the rapid and highly sensitive determination of diclofenac. The modified sensor was characterized by Fourier Transform Infrared (FT-IR) spectroscopy and scanning electron microscopy (SEM). The electrochemical behavior of the screen-printed electrodes was assessed through cyclic voltammetry (CV) in phosphate buffer solution (PBS) and potassium ferrocyanide/potassium ferricyanide solution. The cyclic voltammograms of the electrodes modified with GO and PHEN revealed peaks in PBS related to redox processes of PHEN immobilized in the carbonaceous matrix. Additionally, the active surface area of the electrodes was found to be larger for the modified carbon screen-printed electrode with GO and PHEN, which also showed improved sensitivity to the detection of DCF. The limit of detection (1.53 nM) and the sensitivity of the novel sensor were promising, and these performance characteristics enabled the sensitive detection of DCF in different pharmaceutical products. The selectivity was confirmed to be appropriate based on recovery studies conducted with the pharmaceutical products, which produced values close to 100%.

1. Introduction

Diclofenac, 2-[(2,6-dichlorophenyl) amino]-benzene acetic acid monosodium salt, is a non-steroidal anti-inflammatory drug widely used for pain management and inflammation control in various rheumatic conditions and joint pain [1,2]. There are also preliminary studies on the use of diclofenac (DCF) for the treatment of tuberculosis and urinary tract infections [3,4]. Compared to other non-steroidal medications, DCF is more effective, with fewer adverse reactions [5,6]. DCF is also used to prevent the production of prostaglandins by inhibiting cyclooxygenase, potentially preventing skin cancer and other malignancies [7,8]. The chemical structure of DCF is shown in Figure 1.

Although DCF has many advantages, its improper use can be associated with disadvantages such as extended hepatic metabolism [9,10], neonatal pulmonary hypertension (if ingested when pregnant) [11,12], or stroke [13,14]. Furthermore, the discharge of DCF into the environment leads to poor water quality and poses a threat to fish health [15]. Therefore, various analytical methods for detecting DCF are used as standard methods, such as liquid chromatography [16], chemiluminescence [17], capillary electrophoresis [18], spectrophotometry [19], gas chromatography–mass spectrometry (GC-MS) [20], thin layer chromatography (TLC) [21], and electroanalytical methods. For the development of an analytical method, high selectivity, sensitivity, and minimal interference are required. Even though these methods are used for the identification of compounds in very small amounts, they require laborious sample preparation, extensive time, well-trained technical personnel, and costly materials. In addition to these techniques, electrochemical methods have become important diagnostic tools due to their high sensitivity, simplicity, accuracy, and rapid detection.

In the field of analytical chemistry and sensor technology, the quest for new, more efficient, accurate, and cost-effective methods for the determination of pharmaceutical compounds in complex matrices continues to encourage innovation. Electrochemical sensors play an essential role in analytical chemistry for quantitative and qualitative analysis of electroactive species in samples. They are appreciated for their sensitivity, affordable prices, simplicity of use, and for being ideal materials for the detection of electroactive analytes [22]. Furthermore, screen-printed electrodes (SPE) are gaining increasing attention due to their simplified assembly, system miniaturization, and portability, making them a versatile option for various electrochemical applications. This innovation has the potential to contribute to the understanding of diclofenac’s impact on human health and the environment while facilitating its efficient detection and monitoring.

Electrochemical detection of DCF has seen significant progress in the last few years, especially in the development of sensors with high sensibility and selectivity. Sensors based on metallic nanoparticles, carbon nanomaterials, or conductive polymers reported significant results for the detection of DCF in different types of samples. Graphene-based composites and their derivatives have proven effective in detecting DCF. For instance, Karuppoah et al. [23] have created a sensor using carboxyl-functionalized graphene oxide (GO), which demonstrated remarkable stability. On the other hand, Kimuam et al. [24] have developed an electrode based on Pt and rGO (reduced graphene oxide), which was successfully applied to human urine, and El-Weki et al. [25] have created a carbon paste electrode modified with rGO for the simultaneous analysis of DCF and esomeprazole. However, the development of novel electrochemical sensors based on nanomaterials and electroactive compounds is highly demanded. Among carbonaceous nanomaterials, graphene is of great interest. GO possesses outstanding properties, including excellent electric conductivity, high mechanical strength, flexibility, and a large specific surface area [26]. These characteristics make GO a promising material for various electrochemical applications.

On the other hand, the use of chemically modified electrodes with electroactive compounds is a promising way to develop highly sensitive and selective electrochemical sensors. Phenanthroline (1,10-phenanthroline) is used to modify carbon-based screen-printed sensors, enhancing their electrochemical performances in analyte detection. The modification of screen-printed electrodes with phenanthroline can be achieved by oxidizing the amino groups to form derivatives that attach to the surface of the electrodes, enhancing the electrochemical performances in the detection applications [27]. By functionalizing the electrode surface, phenanthroline increases the electrode’s sensibility and sensitivity, facilitating electron transfer and improving the sensor’s response to analytes such as DCF.

In this work, the electrochemical behavior and analytical performance characteristics of a novel sensor based on a carbon SPE modified with GO and PHEN were studied for the detection of diclofenac. This innovative electrochemical sensor integrates the unique properties of both GO and PHEN, combining their individual sensing benefits to enhance the sensor’s sensitivity and selectivity. The incorporation of GO improves the active area of the sensor, facilitating better interaction with the analyte, while PHEN enhances electron transfer efficiency, further increasing the sensor’s sensitivity. The synergistic effect of these two materials creates a dual-modified sensor, which offers superior performance characteristics, particularly in complex samples such as pharmaceuticals. The novelty of this sensor lies in the combination of GO and PHEN, a strategy that has not been widely explored for DCF detection in previous studies. Furthermore, to date, no other electrochemical sensor for determining diclofenac has been reported using graphene oxide and phenanthroline functionalization.

2. Materials and Methods

2.1. Reagents and Solutions

All the compounds utilized in this study are of analytical purity and were purchased from Sigma-Aldrich (St. Louis, MO, USA). In the preliminary studies, a 0.1 M KCl − 10⁻³ M potassium ferrocyanide/potassium ferricyanide solution was used, prepared by dissolving the adequate amounts of compounds in ultrapure water. The 0.1 M phosphate buffer solution (PBS) of pH 7 was prepared by dissolving calculated amounts of NaH₂PO₄ and Na₂HPO₄ in ultrapure water. The pH was determined using a pH meter and adjusted with NaOH solution or H₃PO₄ if necessary. It was used as a supporting electrolyte in studies for the detection of DCF. The stock solution of 10⁻³ M DCF was prepared by dissolving the pure compound in 0.1 M PBS, pH 7. Diclofenac sodium salt was purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.2. Apparatus and Methods

For the electrochemical measurements, a potentiostat/galvanostat (BioLogic Science Instruments SP-150, Seyssinet-Pariset, France) was used, connected to a computer, and operated using EC-LAB EXPRESS V5.52 software. The experiments were conducted in a 50 mL electrochemical cell (Princeton Applied Research, Oak Ridge, TN, USA) that includes a three electrodes system: an Ag/AgCl reference electrode; a Pt counter electrode; and a working electrode (screen-printed electrode). The working electrode was sequentially C/SPE (carbon SPE), GO/C/SPE (C/SPE modified with graphene oxide), and PHEN/GO/C/SPE (C/SPE modified with graphene oxide and phenanthroline). The screen-printed electrodes (C 110 work in solution) were purchased from Metrohm DropSense (Oviedo, Asturias, Spain). Graphene oxide (powder, 15–20 sheets, 4–10% edge-oxidized) and 1,10-phenanthroline were purchased from Sigma-Aldrich (St. Louis, MO, USA). The modification of commercial C/SPE was carried out with a suspension of GO in ethanol (5 mg/mL) by adding 10 μL in two stages and evaporating the solvent. The GO/C/SPE was further modified with PHEN in ethanol (10 mg/mL) by adding 10 μL in two stages and evaporation of the solvent.

The ultrapure water used for preparing the solutions was obtained using a water purification system, Mili-Q Milipore (Bedford, MA, USA). A UV–Vis Rayleigh UV-1601 spectrometer, connected to a computer and controlled with UVSoftware was used for the spectrophotometric analysis. UV spectra of the DCF solution with a concentration from 5 to 45 µg per 100 mL were measured in quartz cuvettes in the range from 200 to 400 nm.

Fourier Transform Infrared Spectroscopy with Attenuated Total Reflectance (ATR-FTIR) was obtained using a Bruker ALPHA FT-IR spectrometer (Bruker, Ettlingen, Germany). The internal reflection element used was ZnSe. The samples and the background spectra were screened from 4000 to 600 cm⁻¹, and the average of 32 scans at 4 cm⁻¹ resolution was recorded.

The morphological characterization of the chemically modified sensor was conducted using a scanning electron microscope FlexSEM 1000, Hitachi, Tokyo, Japan.

2.3. Pharmaceuticals Product Analysis

For this study, four pharmaceutical products with different formulations and concentrations of DCF, as specified by the manufacturers, were used. The commercial drugs (

Table 1. Pharmaceuticals used in this research and their composition.

Pharmaceutical Product	Administration	Dosage Form	Active Compound	List of Ingredients from the Manufacturer
Diclofenac 50	Oral	gastro-resistant tablets	Diclofenac sodium 50 mg	lactose monohydrate, povidone, corn starch, anhydrous colloidal silica, magnesium stearate, talcum, methacrylic acid ethyl acrylate copolymer 30%, triethyl citrate, titanium dioxide (E171), quinoline yellow (E104), indigotin (E132), simethicone emulsion 30%, and sodium hydroxide.
Diclofenac Duo	Oral	capsules with extended release	Diclofenac sodium 75 mg	microcrystalline cellulose (MCC), polyvidone K25, colloidal silicon dioxide, methacrylic acid-ethyl acrylate copolymer (1:1), propylene glycol, talcum, poly[ethylacrylate-co-methylmethacrylate-co-(2-trimethylammonioethyl methacrylate chloride] 1:2:0,1, poly[ethylacrylate-co-(2-trimethylammonioethyl) methacrylate chloride] 1:2:0.2, dibutyl phthalate, indigotin, titanium dioxide (E171), gelatin, antifoam emulsion, shellac.
Tratul Plus	Oral	gastro-resistant capsules	Diclofenac sodium 50 mg	thiamine hydrochloride (vitamin B1), pyridoxine hydrochloride (vitamin B6), cyanocobalamin (vitamin B12) povidone, methacrylic acid-ethyl acrylate copolymer (1:1), triethyl citrate, talcum, iron oxide red (E172), iron oxide yellow (E172), titanium dioxide (E171), gelatin.
Refen	Injectable	solution	Diclofenac sodium 25 mg/mL	benzyl alcohol, mannitol, sodium hydroxide, propylene glycol, water for injection formulation.

) were bought from a local pharmacy in Galați, România.

2.4. Pharmaceutical Sample Preparation

All the pharmaceutical samples were prepared similarly, based on the pharmaceutical form of the product, as follows: the 50 mg Diclofenac tablet was triturated, and the resulting powder was dissolved in PBS and then brought up to a final volume of 100 mL. The mixture was ultrasonicated for 10 min at room temperature. The resulting solution was then diluted to ensure that the sample concentration fell within the working range for both spectrophotometric and voltametric determinations. For capsules (Diclofenac Duo and Tratul), the content was extracted and homogenized; then, the resulting powder was dissolved in PBS and brought up to a final volume of 100 mL. Refen, which is an injectable solution of DCF, was directly mixed with PBS prior to analysis.

3. Results

3.1. FT-IR and SEM Characterization of the Sensor Surface

FT-IR analysis was used to demonstrate the efficient immobilization of the GO and PHEN on the surface of GO/C/SPE. In Figure S1, the FT-IR spectra of GO/C/SPE and PHEN/GO/C/SPE are shown. As can be seen (red line spectrum), there are several vibrational bands related to functional groups from the graphene oxide structure. In the FTIR spectrum of GO, because of moderate oxidation, GO has a relatively low and broad O–H stretching vibration band at 3400 cm⁻¹, carboxyl C=O stretching band at 1700 cm⁻¹, O–H deformation vibration band at 1400 cm⁻¹, and C–O stretching vibration at 1040 cm⁻¹ [28].

In the case of the PHEN/GO/C/SPE spectrum (blue line), the characteristic vibrational bands are observed. The FTIR spectra show several characteristic peaks: the stretching vibration peak of C=N and C=C double bonds appears at 1580 cm⁻¹ and 1637 cm⁻¹; the skeleton vibration peak appears at 1560 cm⁻¹; CCC bending appears in-plane at 1113 cm⁻¹; CCC bending appears in-lane; HCC bending appears in-plane at 1179 cm⁻¹; HCC bending appears in-plane at 1232 cm⁻¹, and the out-plane bending vibration peak of C–H bond appears at 751 cm⁻¹ [29].

The surface morphology was analyzed using SEM, and the image obtained for PHEN/GO/C/SPE is presented in Figure S2.

The SEM image reveals 2D nanosheet morphologies with wrinkled and folded textures, occasionally exhibiting irregular edges, rough surfaces, and crumpling due to scrolling [30]. Only small formations of PHEN can be observed on the surface of the GO 2D nanosheet, likely due to the diffusion of the solution on the GO during evaporation.

Therefore, the FT-IR and SEM methods were effective in demonstrating the presence of GO and PHEN on the sensor surfaces, as well as the nanostructure of the sensitive element.

3.2. Preliminary Studies in PBS and Ferrocyanide/Ferricyanide Redox Probe

The preliminary studies examined the electrochemical behavior and characteristics of three types of electrodes—C/SPE, GO/C/SPE, and PHEN/GO/C/SPE—both in the electrolyte solution and a reference redox probe solution. Firstly, the behavior of the electrodes in 0.1 M PBS was studied, followed by their behavior in the electroactive solution containing 10⁻³ M K₄[Fe(CN)₆]/K₃[Fe(CN)₆] − 0.1 M KCl.

Initially, the potential range to be employed was optimized. In a wide potential range from −0.8 to 1.2 V, the electrode signals were slightly unstable, showing a small cathodic peak of low intensity, likely associated with the reduction in water, as reported by other researchers [31,32]. Therefore, an increase in the negative limit of the potential was necessary. After the optimization stage, the applied potential range was set between −0.4 and 1.0 V, resulting in stable signals for all electrodes. The scan rate used was 0.1 V·s⁻¹, which was an appropriate value for the screen-printed electrodes, which ensured a low background current and enabled a clear observation of the redox processes at the electrode’s active surface.

The voltametric curves of the C/SPE and GO/C/SPE sensors in 0.1 M PBS did not show redox peaks, confirming the high quality of the materials used in manufacturing the electrodes. The background current was the lowest for the GO/C/SPE electrode, which could be attributed to the use of this nanomaterial on the active surface, facilitating the electrochemical processes. When the PHEN/GO/C/SPE sensor was used, peaks corresponding to the redox process of PHEN were observed. One anodic peak appeared at 0.097 V, one reduction peak at −0.007 V, and another at −0.305 V. These electrochemical processes are shown in Figure 2, in agreement with previously reported results [33].

In the next stage, the electrochemical behavior of the electrodes immersed in potassium ferrocyanide–ferricyanide solution (10⁻³ M K₄[Fe(CN)₆]/K₃[Fe(CN)₆] − 0.1 M KCl) was studied (Figure 3). Each electrode exhibited two peaks (an anodic and a cathodic peak) associated with the redox processes of the ferrocyanide/ferricyanide ions on the electrode surface. At the C/SPE, the cyclic voltammogram of ferrocyanide/ferricyanide ions (blue line) showed a pair of redox peaks, with the anodic peak potential at 0.470 V and the cathodic peak potential at 0.139 V in 0.1 M KCl. For the GO/C/SPE, a pair of redox waves of ferrocyanide/ferricyanide ions (red line) were observed. The anodic peak potential was located at 0.416 V and the cathodic peak potential at 0.184 V, respectively. Furthermore, at the PHEN/GO/C/SPE, the cyclic voltammogram of ferrocyanide/ferricyanide ions (green line) showed one anodic peak at 0.419 V and the cathodic peak at 0.201 V. Additionally, in the case of PHEN/GO/C/SPE, an additional cathodic peak (at a potential of −0.245 V) was observed, related to the PHEN immobilized on the sensor’s active surface (Figure 3).

The electrochemical parameters obtained from cyclic voltammograms are detailed in

Table 2. Electrochemical parameters obtained from voltammograms of sensors immersed in 10⁻³ M ferrocyanide–ferricyanide—0.1 M KCl solution.

Electrode	1Epa (V)	2Epc (V)	3E1/2 (V)	4∆E (V)	5Ipa (mA)	6Ipc (mA)	Ipc/Ipa
C/SPE	0.470	0.139	0.304	0.331	0.0159	−0.0150	0.943
GO/C/SPE	0.416	0.184	0.300	0.232	0.0091	−0.0086	0.945
PHEN/GO/C/SPE	0.419	0.201	0.310	0.218	0.0186	−0.0167	0.897

¹E_pa, ²E_pc—the potential of anodic and cathodic peaks, respectively; ³E_1/2—the half-wave potential; ⁴∆E—the difference between anodic peak and cathodic peak potentials; ⁵I_pa, ⁶I_pc—the current corresponding to anodic and cathodic peaks, respectively.

.

Both the anodic and the cathodic peak intensities are higher for the PHEN/GO/C/SPE sensor, with a difference of 2.7 μA compared to C/SPE in the anodic scan, and nearly double compared to GO/C/SPE, as shown in Figure 3 and Table 2. This indicates that the redox process of the ferro-ferricyanide ions is enhanced by the presence of phenanthroline, which increases the rate of electron transfer.

The half-wave potential (E_1/2) depends on the nature of the electroactive species, as well as the composition and pH of the electrolytic solution [34,35]. In this case, E_1/2 values are similar, suggesting that the electrodes exhibit comparable electrochemical behavior. Considering the minimum value of ΔE of 0.218 V for PHEN/GO/C/SPE, there is a tendency toward a more stable and efficient electron transfer behavior, indicating a redox reaction closer to reversibility compared to the other sensors. The I_pc/I_pa ratio was optimal for all three sensors, with values close to the ideal value of 1.

To estimate the active surface area of the sensors, cyclic voltammograms were recorded in a 10⁻³ M potassium ferrocyanide/potassium ferricyanide—10⁻¹ M potassium chloride solution at different scan rates ranging from 0.1 to 1 V·s⁻¹ (Figure S4). For all three electrodes immersed in the electroactive solution, both anodic and cathodic currents increased with the scan rate. By analyzing the relationship between the anodic currents and the square root of the scan rates, a linear correlation was observed for all electrodes (Figure S4). Therefore, the electrochemical processes of ferrocyanide ions are controlled by the diffusion process [35].

The electroactive surface area of the electrodes was calculated using the Randles–Sevcik equation, considering the diffusion coefficient of ferrocyanide, D = 7.26 × 10⁻⁶ cm² × s⁻¹ [36]:

$${\mathrm{I}}_{\mathrm{p}\mathrm{a}}=\mathrm{268,600}\times n^{3/2}\times \mathrm{A}\times D^{1/2}\times C\times v^{1/2}$$

(1)

where I_pa is the anodic current (A); n is the number of electrons transferred in the redox process; A is the area of the active surface of the electrode (cm²); D is the diffusion coefficient (cm²·s⁻¹); C is the concentration (mol·cm⁻³), and v is the scan rate (V·s⁻¹).

The PHEN/GO/C/SPE sensor exhibits an active surface area approximately eight times greater than the geometric surface area of the sensor, while the GO/C/SPE has an active surface area only three times larger than its geometric surface area (

Table 3. Active area and geometric area of the surface of the electrodes used in this analysis.

Electrode	Active Area (cm2)	Geometric Area (cm2)	R2
C/SPE	0.635	0.1256	0.9998
GO/C/SPE	0.3908		0.9946
PHEN/GO/C/SPE	1.0258		0.9973

). This increased active area, and, therefore, the increased sensitivity of PHEN/GO/C/SPE, is due to the electrochemical properties of PHEN immobilized on the surface, which facilitate and enhance the rate of electron transfer.

3.3. Electrochemical Detection of Diclofenac in Aqueous Solution

Cyclic Voltammetry (CV) is an efficient electrochemical technique for the detection of diclofenac, providing detailed information about the electrochemical behavior of this compound and allowing for the evaluation of the sensitivity and selectivity of electrochemical sensors. This method is well-regarded for its sensitivity and selectivity, making it valuable for monitoring processes occurring on the surface of electrodes [37]. Furthermore, CV can be used for the quantification of diclofenac in different pharmaceutical products based on a calibration curve.

The detection of DCF (10⁻³ M solution, 0.1 M PBS as the supporting electrolyte, pH = 7) with cyclic voltammetry (CV) with three electrodes—C/SPE, GO/C/SPE and PHEN/GO/C/SPE—was studied. The cyclic voltammograms were recorded at a scan rate of 0.1 V·s⁻¹ within the optimized potential range of −0.3 to 1.0 V. At a wider potential range, the stability of the response was satisfactory, especially for PHEN/GO/C/SPE. The results obtained are shown in Figure 4.

In this study, using all the electrodes, a well-defined peak was observed at a potential of approximately 0.670 V during the first voltametric scan without a cathodic counterpart, suggesting the formation of 5-hydroxydiclofenac through an irreversible electrochemical mechanism. In successive scans, the unstable oxidation product of DCF accumulates on the surface of the electrode, explaining the appearance of a pair of peaks associated with a quasi-reversible redox process of 5-hydroxydiclofenac. The entire electrochemical mechanism involves the transfer of two electrons and two protons, similar to those reported in previous studies [38,39], and is depicted in Figure 5.

The literature suggests that DCF can be electrochemically oxidized through various redox mechanisms, depending on the electrode used and the experimental conditions. For example, Goyal et al. [40] described an oxidation mechanism at pH 7.2 on a pyrolytic graphite electrode, where DCF is irreversibly oxidized at 5-hydroxydiclofenac by a transfer of two electrons and two protons, with an oxidation peak at approximately 0.66 V. In the cathodic cycle, 5-hydroxydiclofenac is reduced to diclofenac 2,5-quinone imine, which is oxidized in the next cycle. On the other hand, Cid-Cerón et al. [41] proposed a reversible mechanism using carbon paste electrodes, where the reaction involves the formation of a radical intermediate and hydrolysis to the final products, 2,6-dichloroaniline and 2-hydroxyphenylacetic acid. Similarly, Aguilar-Liras et al. [42] confirmed this mechanism for the oxidation of DCF at pH 7.0 using graphite-modified electrodes, observing both a quasi-reversible process and an irreversible redox process.

The position and intensity of the peaks vary depending on the composition, structure, and properties of the materials used in the fabrication of the SPCEs. Notably, the use of the PHEN/GO/C/SPE results in higher signal intensity and a lower oxidation potential for DCF, suggesting the electrocatalytic effect of PHEN, which increases sensitivity and reduces activation energy. The low potential value indicates a rapid electron transfer process occurring at the active surface of the electrode [43].

The influence of the solution pH on the oxidation of DCF was examined in a 0.1 M phosphate buffer solution (PBS) within the pH range of 4.0 to 9.0. It was observed that the current intensity progressively increased up to around pH 7 (Figure 6a). At a more alkaline pH value, the current intensity decreased. Similarly, as the pH increased, a shift in potential toward less-positive values was observed (Figure 6b). Therefore, the solution pH was set to 7, which is optimal, as reported in other studies [24,44,45].

To study the influence of the scan rate on the electrochemical responses of the sensors, the electrochemical behavior of three sensors—C/SPE, GO/C/SPE, and PHEN/GO/C/SPE—was examined in a DCF solution with a concentration of 10⁻⁴ M (PBS 10⁻¹ M, pH 7), using scan rates ranging from 0.1 to 1.0 V·s⁻¹. The results showed that anodic peaks increased with the scan rate (Figure S3). For each of the three sensors, a well-defined linear dependence between the anodic peak current (E = 0.688 V) and the scan rate was observed, indicating that the oxidation process of DCF at the electrode surface was controlled by its adsorption on the active surface of the sensor.

From these results, it is possible to quantify the coverage of the active surface with DCF using Laviron’s equation, which describes the relationship between anodic current and scan rate [43]. Laviron’s equation is as follows:

$${\mathrm{I}}_{\mathrm{p}\mathrm{a}}={\displaystyle \frac{{\mathrm{n}}^2{\mathrm{F}}^2\mathsf{\Gamma }\mathrm{A}\mathrm{v}}{4\mathrm{R}\mathrm{T}}}$$

(2)

where I_pa represents the anodic peak current; n is the number of transferred electrons; F is the Faraday’s constant; Γ is the degree of coverage of the electrode surface with DCF; A is the surface area of the electrode; v is the scan rate; R is the ideal gas constant, and T is the absolute temperature.

Based on this relation and the analysis of the linear dependence between the anodic current and the scan rate, the values of Γ were calculated for each sensor, as reported in

Table 4. The linear regression equation of electrochemical sensors (R²—coefficient of determination; ᴦ—degree of coverage of the electrode surface; I_pa—anodic peak current).

Sensor	Linear Equation	R2	ᴦ (mol/cm2)	Epa (V)
C/SPE	Ipa = 0.0000085·v + 0.0045	0.9900	3.265 × 10−12	0.676
GO/C/SPE	Ipa = 0.00001·v + 0.0038	0.9925	6.242 × 10−12	0.666
PHEN/GO/C/SPE	Ipa = 0.00001·v + 0.0053	0.9904	2.401 × 10−11	0.688

.

The results obtained for each sensor reveal that the oxidation process is faster and more pronounced for the PHEN/GO/C/SPE, suggesting more efficient adsorption of DCF on its active surface and resulting in superior electroanalytical performance for DCF detection.

3.4. Calibration Curve Registration

Analysis of previous experimental data indicates that the PHEN/GO/C/SPE sensor exhibits superior performance, which can be attributed to the modification with phenanthroline, enhancing its interaction with DCF. Cyclic voltammograms were recorded using the PHEN/GO/C/SPE sensor to obtain the calibration curve. In this stage, volumes of 5 to 50 μL of 10⁻⁴ M DCF solution (PBS 10⁻¹ M, pH 7.0) were sequentially added while the mixture was continuously stirred using a magnetic stirrer. The concentration range studied was 0.01–1.75 μM. The results are shown in Figure 7.

As shown in Figure 7, the intensity of the anodic peak current increased proportionally with the DCF concentration, demonstrating the efficient and stable electrocatalytic capability of the PHEN/GO/C/SPE sensor.

The calibration curve was obtained by representing the anodic peak current as a function of DCF concentration. The results are shown in Figure 8a,b. The current response was linear within the DCF concentration range from 0.01 to 0.07 μM.

Using the linear equation, the limits of detection (LOD) and the limits of quantification (LOQ) were calculated in accordance with the equations LOD = 3σ/m (where σ is the standard deviation, and m is the slope of the calibration curve) and LOQ = 10σ/s [31]. The calculated values are presented in

Table 5. Limit of detection and limit of quantification.

Sensor	Linear Equation	R2	LOD (μM)	LOQ (μM)
PHEN/GO/C/SPE	Ipa (μA) = 0.1559·c(μM) + 1.6	0.9968	0.00153	0.00508

.

The PHEN/GO/C/SPE exhibits superior analytical performance compared to other sensors reported in the literature, as demonstrated by the comparison of LOD and LOQ, shown in

Table 6. Summary of sensors from the literature for DCF detection.

Sensor/Sensitive Material	Method	Linearity Range (μM)	LOD (μM)
MIP [46]	CV	0.1–10	3.5
Graphene/Gadolinium oxide [47]	CV	5.86–66.6	0.028
Au-PtNPs/f-MWCNTs/AuE [48]	DPV	0.5–1000	0.3
MoS2 Nanosheets Modified Graphite [49]	CV	0.05–600	0.03
CeO2-SPCE [50]	DPV	0.05–1 2–250	0.4
Graphene/MWCNT/copper-nanoparticle [51]	DPV	17.41–206.45	0.057
Pt nanoflowers/reduced GO [24]	DPV	0.1−100	0.04
MWCNTs [52]	DPV	0.047–12.95	0.017
MWCNTs/Cu(OH)2 NPs/IL/GCE [53]	DPV	0.18–119	0.04
GO/COOH [23]	CV, LSV	1.2–400	0.09
Ru/TiO2 [54]	CV	0.291	11.48
SWCNT modified EPPGE [55]	SWV	0.02–1.5	0.02
Graphene—Co3O4/SPGE [56]	DPV	0.02–575	0.007
Carbon modified with graphene oxide and phenanthroline—this work	CV	0.01–0.07	0.00153

. Their improved results can be attributed to the presence of phenanthroline, which enhances the interaction with the analyte.

3.5. Diclofenac Detection in Pharmaceutical Products

The PHEN/GO/C/SPCE was used for the determination of DCF in various pharmaceutical formulations by recording cyclic voltammograms in solutions of the pharmaceutical products dissolved in 0.1 M PBS of pH 7.0. The procedure involved dissolving a sample of the compound (one capsule, one tablet), either triturated or in its original state, in 1000 mL of distilled water. The solution was then sonicated for 10 min and allowed to settle for 1 h to facilitate decantation. A volume of 100 mL was taken from the supernatant of each solution for analysis. Three replicate measurements were performed for each pharmaceutical product, where 50 μL, 100 μL, and 150 μL aliquots of the supernatant were added to 50 mL of 0.1 M PBS (pH 7.0) for analysis.

The cyclic voltammograms of PHEN/GO/C/SPCE immersed in solutions of pharmaceutical products are shown in Figure 9.

It can be observed, in all cases, that the characteristic peaks of DCF appear at the same potentials as those observed for the pure compound. Therefore, the influence of excipients on the sensor signal is minimal. Based on these results, the DCF content in the pharmaceutical samples was determined using the anodic peak current (E = 0.688 V) and the calibration equation. The results are summarized in

Table 7. Determination of diclofenac concentration in real samples by means of UV spectrophotometric method and electrochemical method.

Pharmaceutical Product	Diclofenac Sodium Content
	Indicated by Producer	Conc. of the Sample (M)	Determined by UV (M) ± RSD (%)	Recovery (%)	Determined by Sensor (M) ± RSD (%)	Recovery (%)
Diclofenac 50 (tablet)	50 mg/caps	1.69 × 10−6 3.38 × 10−6 5.12 × 10−6	1.65 × 10−6 ± 0.01 3.13 × 10−6 ± 0.03 4.20 × 10−6 ± 0.02	97.63 92.60 82.03	1.58 × 10−6 ± 1.19 2.95 × 10−6 ± 0.88 4.14 × 10−6 ± 0.97	93.49 87.47 80.85
Diclofenac Duo (capsule)	75 mg/caps	2.53 × 10−6 5.06 × 10−6 7.59 × 10−6	2.43 × 10−6 ± 0.11 4.20 × 10−6 ± 0.08 6.23 × 10−6 ± 0.18	96.04 83.00 82.08	2.46 × 10−6 ± 0.19 3.95 × 10−6 ± 0.77 6.32 × 10−6 ± 0.47	97.23 78.06 83.26
Tratul Plus (capsule)	50 mg/caps	1.69 × 10−6 3.38 × 10−6 5.12 × 10−6	1.47 × 10−6 ± 0.21 4.27 × 10−6 ± 0.19 5.40 × 10−6 ± 0.09	86.98 126.33 105.46	1.50 × 10−6 ± 0.36 4.26 × 10−6 ± 0.71 5.73 × 10−6 ± 0.26	88.75 126.03 111.91
Refen (injectable solution)	75 mg/3 mL	2.35 × 10−6 4.70 × 10−6 7.05 × 10−6	2.00 × 10−6 ± 0.07 4.19 × 10−6 ± 0.12 6.20 × 10−6 ± 0.13	85.10 89.15 87.94	2.02 × 10−6 ± 0.82 4.23 × 10−6 ± 0.65 6.25 × 10−6 ± 0.36	85.95 90.00 85.46

.

The results were validated at the laboratory level using a spectrophotometric method in the UV range. Solutions of different concentrations of DCF (5–45 μg/100 mL) were prepared, and spectra were recorded in the 200–400 nm range, revealing a maximum absorbance at 276 nm (Figure S5).

These results are in agreement with the data published in the scientific literature [57,58]. The linear calibration regression model was obtained from the graphical representation of the absorbance at 276 nm as a function of the DCF concentration in the analyzed solution, obtaining the linear equation A = 0.0382·c, R² = 0.992.

The quantity of DCF in the analyzed pharmaceutical products was calculated using the absorbances of the samples and the calibration equation, considering the dilution of the samples and the absorbance at 276 nm. The results are presented in Table 7.

The electrochemical method generally provides satisfactory results for most samples, with only minor exceptions, which may be attributed to the formulation or excipients present in the pharmaceutical products.

3.6. Stability, Reproducibility, Repeatability, and Interference Studies

The stability of the C/PHEN/SPE was studied by measuring 100 repeated cyclic voltammograms in a DCF 10⁻⁶ M solution. The results demonstrated that the sensor maintained its stability throughout the measurements, with no significant changes in the anodic peak current.

The variation in sensor response when determining DCF in solutions of the same concentration was also investigated. After removing the sensor from the solution, rinsing it with PBS, and repeating the cyclic voltammogram, the difference in current intensities was less than 2%, demonstrating the reproducibility of the PHEN/GO/C/SPE sensor determination.

A few chemical species encountered in pharmaceuticals were utilized for interference studies, and the findings are summarized in

Table 8. Interference of chemical species in the detection of diclofenac (10⁻⁶ M) concentration.

Interfering Compound	Concentration (M)	Recovery (%)	Coefficient of Variation (%)	Tolerance Limit (M)
Acetaminophen	10−5	98.73	2.5	2 × 10−4
Phenylbutazone		99.75	3.0	5 × 10−4
Ibuprofen		101.32	1.8	10−4

.

The recovery value was calculated with the following equation:

$$\mathrm{Recovery}={\displaystyle \frac{measured concentration value}{sample concentration value}}\times 100$$

The coefficient of variation (RSD) was calculated using the following formula:

$$RSD={\displaystyle \raisebox{1ex}{$\sigma $}\!\left/ \!\raisebox{-1ex}{$\mu $}\right.}\times 100$$

where µ is the mean, determined based on three repeated measurements of DCF concentration, and σ is the standard deviation, calculated from these three values to assess the precision of the obtained results.

The tolerance limit was defined as the maximum concentration of the interfering compound that resulted in an approximately ±5% relative error in the determination.

The recovery values and the coefficient of variation were determined by comparing the measured DCF concentrations with the values provided by the manufacturer and evaluating the sensor’s response in the presence of various interferents. The recovery values reflect the efficiency of the detection method, while the coefficient of variation indicates the precision of the measurements. The tolerance limits were set based on the concentration of interferent that resulted in a coefficient of variation of 5% (Table 8). These parameters were calculated to assess the sensor’s performance and ensure the reliability of the obtained results.

The results in Table 8 show that the anodic peak values associated with DCF do not change significantly, even with the addition of interferents to the analyte solution. The PHEN/GO/C/SPE exhibited excellent selectivity, as both the anodic peak potential and current remained almost unchanged in the presence of various interfering species.

4. Conclusions

In summary, a new chemically modified sensor was developed and successfully applied for the determination of diclofenac in pharmaceutical products. The electrochemical characterization of the sensors at different stages of modification highlights the significant role of material modification in enhancing sensitivity. GO was used to enhance the rate of electron transfer and increase the active surface area of the sensor. The electrocatalytic effect of phenanthroline improves sensitivity, increases process reversibility, and reduces the activation energy of the DCF redox electrochemical processes. PHEN/GO/C/SPE showed excellent responses in DCF determination and demonstrated good linearity in kinetic analysis. During the calibration process, the sensor proved to be ultra-sensitive for DCF detection, with a linearity range from 0.01 to 0.07 μM and a detection limit of 1.53 nM. Additionally, the results from the novel sensor demonstrated satisfactory repeatability, reproducibility, and selectivity, with minimal interference, suggesting that the sensor is a reliable and effective analytical tool for the determination of DCF in various pharmaceutical forms. Its applicability in the quantitative analysis of DCF in pharmaceutical products was successfully demonstrated. Overall, the development and application of electrochemical methods for the determination of active compounds in various samples, including pharmaceutical ones, is favored due to their rapid response, high selectivity and sensitivity, improved versatility, cost-effectiveness, and low toxicity, making this method environmentally friendly.