Simultaneous Electrochemical Determination of Dopamine, Acetaminophen, and Caffeine with a PVP/rGO-Modified Electrode

Abstract

The simultaneous and cost-effective determination of Dopamine (DA), Acetaminophen (APAP), and Caffeine (CAF) could be useful in several areas such as pain management, clinical diagnostics, and pharmaceutical quality control. In this study, reduced graphene oxide (rGO) was synthesized from graphite and graphene oxide (GO) using ascorbic acid as reducing agent. The rGO was characterized using FT-IR spectroscopy, Raman spectroscopy, scanning electron microscopy, and thermogravimetry. rGO and polyvinylpyrrolidone (PVP) were then combined to modify a glassy carbon electrode (rGO-PVP/GCE), which was studied using cyclic voltammetry, electrochemical impedance spectroscopy, and square-wave voltammetry (SWV). The modified electrode rGO-PVP/GCE exhibited favorable electron transfer kinetics and electrocatalytic activity in the oxidation of DA, APAP, and CAF compared to GO/GCE, PVP/GCE, and rGO/GCE. The SWV results showed three distinct anodic peaks at 0.44, 0.63, and 1.49 V (vs Ag/AgCl 3 M KCl), corresponding to the oxidation of DA, APAP, and CAF, respectively. The responses showed good linearity in the investigated concentration ranges and the limits of detection for DA, APAP, and CAF were 0.81, 0.16, and 19.6 μM, respectively. Finally, the proposed sensor was applied to simultaneously determine DA, APAP, and CAF in artificial urine, a pharmaceutical syrup, and an energy beverage.

1. Introduction

Acetaminophen, Caffeine, and Dopamine are three substances involved in pain management. Acetaminophen (APAP), or Paracetamol, is a commonly used analgesic and antipyretic medication. Dopamine (DA) is a neurotransmitter that plays a critical role in mood and various bodily systems. In Parkinson’s disease, for instance, the concentration of DA in extracellular fluid is a crucial diagnostic factor. On the other hand, Caffeine (CAF) is a small molecule found in many plant products and beverages, and has several important pharmacological effects, including central nervous system stimulation and boosting of DA levels. APAP is sometimes combined with CAF to amplify its analgesic effects, making the combination particularly effective to treat headaches and migraines. Simultaneously measuring DA, APAP, and CAF may help monitor patients who may be using the combination of APAP and CAF to ensure efficacy and safety. These compounds can be monitored in patient urine, − plasma, serum and saliva. , Several methods, including spectrophotometry, liquid chromatography, chemiluminescence, and electrochemical methods have been employed to determine these compounds, often individually. −

Multianalyte electrochemical sensors offer a cost-effective approach for simultaneous detection of multiple targets; however, identifying materials that enable high sensitivity and selectivity remains a significant challenge. Previous studies have explored modified electrodes incorporating quantum dots, metal nanoparticles, or conducting polymers to improve sensitivity for multianalyte detection. − While these strategies can enhance performance, they often face some limitations such as poor reproducibility, complex fabrication procedures, and signal interference in real-sample applications. , The integration of carbon-based nanomaterialsparticularly graphene and reduced graphene oxide (rGO) as surface modifiers has emerged as a promising strategy due to their excellent electrical conductivity, large surface area, and tunable surface chemistry. , Graphene oxide (GO) can be produced at lower cost than graphene, and its oxygen-containing groups make it dispersible in water. However, its conductivity is generally diminished compared to pure graphene. To address this shortcoming, GO can be reduced into rGO by using reducing agent to fine-tune its properties.

Although rGO has favorable electrocatalytic properties, in many solvents, rGO nanosheets rapidly aggregate due to van der Waals interaction and strong π–π stacking. This can restrict its application for electrode modification. To overcome this disadvantage, several strategies have been developed. − Among them, polyvinylpyrrolidone (PVP) is a hydrophilic polymer that interacts with oxygen-containing functional groups on the rGO nanosheets through hydrogen bonding and van der Waals interactions. These interactions prevent π–π stacking and aggregation of rGO sheets, promoting the formation of a more homogeneous dispersion. During electrode modification, this well-dispersed suspension enables the formation of a uniform and cohesive film. , In addition, PVP exhibits strong adsorption affinity toward phenolic compounds, which can be advantageous for the detection of analytes bearing phenolic-like structures.

This study aims to develop a sensitive electrochemical sensor for the simultaneous detection of dopamine, acetaminophen, and caffeine, leveraging the synergistic effect of polyvinylpyrrolidone (PVP) and reduced graphene oxide (rGO) to enhance electrochemical oxidation. The objective is to achieve high sensitivity and selectivity for these three analytes (DA, APAP, and CAF), which may be found together in biological samples and pharmaceutical formulations.

2. Materials and Methods

2.1. Reagents and Solutions

Reagents in this study include Acetaminophen (C₈H₉NO₂, Acros Organics), Dopamine, Caffeine, polyvinylpyrrolidone (PVP, Carlo Erba), and potassium ferricyanide (K₃[Fe­(CN)₆], Acros Organics). Graphite was obtained from ChemPUR (Karlsruhe, Germany). Alumina (Al₂O₃), disodium hydrogen phosphate, potassium dihydrogen phosphate, and potassium chloride were obtained from QrëC (Auckland, New Zealand). All chemicals used in this work were of analytical grade. All aqueous solutions were prepared with deionized (DI) water. An artificial urine product (Sigmatic Urine Diluent) was utilized as a substitute for a biological sample. The drug and energy drink samples, which were purchased commercially, were diluted in acetate buffer without undergoing any pretreatment.

2.2. GO and rGO Synthesis and Characterization

In this study, GO was prepared using a modified Hummers’ method. Initially, 2 g of natural graphite powder were dispersed in a round bottle flask containing 68 mL of concentrated H₂SO₄, 1.5 g of NaNO₃, with thermometer and a gas trap. Gradually, 9 g of KMnO₄ was added to the mixture over a period of 30 min, while maintaining the reaction temperature below 35 °C by placing the reaction mixture on an ice water bath. The mixture was then allowed to cool for 2 h in the ice water bath, followed by keeping it below 35 °C for 5 days. Next, 40 mL of 0.1 M H₂SO₄ was added to the mixture, and the resulting solution was stirred overnight. The unreacted KMnO₄ was then removed using 6 mL of 30%H₂O₂, and the final mixture was centrifuged at 4000 rpm for 10 min. The collected graphite oxide precipitate was then redispersed in H₂O and centrifuged again. This procedure was repeated 20 times to raise the pH, and the outer solution was monitored for UV–VIS absorbance until the absorbance became nearly zero. Finally, the graphene oxide precipitated slurry was stored at – 1 °C.

Ascorbic acid is a low-cost, nontoxic reducing agent that can reduce GO under mild conditions, resulting in a higher degree of reduction compared to other agents, and producing a more conductive and stable material. The reduced graphene oxide was prepared by using ascorbic acid as the reduction agent according to Silva et al. Initially, a slurry of 100 mg of graphene oxide (GO) was prepared in 100 mL of distilled water by sonication. Later, 100 mg of ascorbic acid was added to the slurry. The pH of the medium was raised to approximately 10 by adding 28% NH₃ solution to promote colloidal stability through electrostatic repulsion. The mixture was stirred at 65 °C for an hour. The obtained suspensions were filtered using cellulose acetate membrane filter papers, washed with abundant DI water and dried at 80 °C for 2 h. ,

The chemical functionalities present in GO and rGO were examined using a Spectrum 100 FT-IR spectrometer (PerkinElmer, USA). The surface characteristics of the GCE with various modifications were examined by a field-emission scanning electron microscope (Zeiss AURIGA FE-SEM/FIB/EDX, Carl Zeiss Microscopy GmbH, Jena, Germany) using 1 keV acceleration and a 30 μm aperture. Raman spectra of graphite powder, GO, and rGO film were acquired with a Raman Microscope (Bruker Senterra), utilizing an argon laser of 532 nm with 5 mW power. Thermogravimetric analyzer (TGA, Mettler Toledo model STAR^e) analyses were performed with a heating rate of 10 °C/min under the nitrogen atmosphere.

2.3. Electrode Modification

To prepare the glassy carbon electrode (GCE, 3 mm diameter) surface, it was first polished with fine emery paper and chamois leather containing alumina (Al₂O₃) slurry with decreasing particle sizes of 5, 1, and 0.5 μm. The electrode was then sonicated in DI water and left to dry in air. For the modification of the GCE, 1 mg of rGO and 0.25 mg of PVP were dispersed in 1 mL of water via sonication for 2 h until completely dissolved. A suspension of a mixture of rGO and PVP was then created. The rGO/GCE, PVP/GCE, and PVP-rGO/GCE electrodes were prepared by separately dropping 2 μL of each suspension onto the surface of a clean GC electrode and drying them under an IR lamp for 5 min. After each measurement, the modified electrodes were regenerated by washing them thoroughly with DI water and sonication for approximately 2 min to remove any adsorbate from the electrode surface, providing a clean surface for subsequent experiments.

2.4. Instrumental Measurements

The voltammetric experiments were carried out at room temperature using a potentiostat (Autolab PGSTAT204 with Nova 2.1 software package, Utrecht, The Netherlands) and a three-electrode setup, which included a Ag/AgCl 3 M KCl reference electrode, a Pt sheet (1 × 1 cm) counter electrode, and a glassy carbon electrode (GCE) working electrode. Electrochemical impedance spectroscopy was conducted using a PalmSens 4 potentiostat/galvanostat controlled by PSTrace 5.10 software (PalmSens, Houten, The Netherlands), and the same reference and counter electrodes were used for the impedance measurements of the GCE and the modified GCE. The pH number of the buffer solution were determined with a calibrated pH meter (Mettler Toledo, Columbus, OH, USA).

3. Results and Discussion

3.1. Characterization of Materials

FT-IR spectroscopy was used to characterize oxygen functional groups and bonding configurations in GO and rGO (Figure A). Both samples show a strong, broad band between 3005 and 3450 cm^–1 due to O–H stretching, confirming the presence of oxygen groups and the oxidation of graphite. After chemical reduction, the CO stretching bands of GO (at 1620 and 1730 cm^–1) decrease significantly. Additionally, the C–O–H bending (1415 cm^–1) and C–O–C stretching (1226 cm^–1) bands disappear in rGO, indicating that most oxygen functionalities have been removed. , However, rGO shows a more intense and shifted C–O stretching band (at 1055 and 986 cm^–1), suggesting the reduction of carbonyl groups and the formation of hydroxyl groups. When incorporating PVP to rGO, the CO band is displaced slightly from 1620 to 1650 cm^–1 because of the carbonyl group in PVP. rGO-PVP shows new peaks at 1422 cm^–1 and 1285 cm^–1 attributed to C–N stretching vibrations of PVP’s aliphatic chains. rGO-PVP also shows a C–H stretching band at 2850–2950 cm^–1 not visible in rGO and the strong band at 1055 cm^–1 (C–O stretching) of rGO is shifted to 1060 cm^–1. Overall, the IR spectrum of rGO-PVP clearly differs from that of pure rGO, confirming that PVP has been incorporated and interacts with the rGO sheets.

1.

(A) FT-IR spectra, (B) Raman spectra and (C) TGA profiles of graphite powder (black), GO (red), rGO (blue), PVP (green) and rGO-PVP (purple).

Figure B shows the Raman spectrum of graphite powder, which exhibits an intense G band at 1580 cm^–1 and a weaker D band at 1355 cm^–1. The second-order region of the graphite powder spectrum shows an intense 2D band at 2718 cm^–1 and a weak shoulder on the G band at 1618 cm^–1. The Raman spectrum of GO shows an increased I _D/I _G ratio from 0.25 to 1.00 compared to the graphite powder. The 2D band in the GO spectrum is broader and has a smaller relative intensity compared to the G band. These features indicate structural disorder in the GO carbon lattice resulting from the oxidation process. The intensity ratios of the D and G bands (I _D/I _G) were estimated to be 0.99, 0.95, and 0.93 for GO, rGO and rGO-PVP, respectively. The lower I _D/I _G ratio of rGO compared to GO indicates the removal of 6-fold ring distortions in the carbon lattice during the reduction process, resulting in a graphitic state with defects. The incorporation of PVP further lower this ratio, suggesting mild additional disorder from PVP. The band at 2880 cm^–1 is attributed to the symmetric stretching vibration of CH₂ groups within the PVP backbone and it is absent in the spectrum of pure rGO. These findings are consistent with previous research. ,

Figure S1 presents FE-SEM images of GO, rGO and rGO-PVP. In Figure S1A, the GO surface appears wrinkled and somewhat uneven. At higher magnification, the layers and folds are more apparent, reflecting the multilayered nature of GO. By contrast, the rGO images (Figure S1B) exhibit a smoother surface with fewer wrinkles and defects, resulting from the removal of oxygen groups and a partial restoration of the graphene structure. Compared to rGO, rGO-PVP exhibits more distinct and isolated sheets with a smoother and more uniform structure (Figure S1C). This evidence that PVP helps to disperse the rGO sheets.

Thermogravimetric analysis (TGA) was performed to evaluate the thermal stability and decomposition behavior of each material, including the rGO-PVP composite. The TGA curves are presented in Figure C. Graphite exhibited minimal weight loss in the temperature range tested, reflecting its high thermal stability. By contrast, graphene oxide (GO) showed the lowest thermal stability, with a pronounced weight loss between 100–150 °C. This loss can be attributed to the decomposition of labile oxygen-containing functional groups, such as hydroxyl, epoxy, and carboxyl groups. Reduced graphene oxide (rGO) exhibited a significantly improved thermal stability compared to GO. It experienced a gradual weight loss up to 200 °C, corresponding to the elimination of residual oxygen functionalities and adsorbed moisture. At higher temperature, the weight loss stabilized. This enhanced thermal resilience stems from the partial restoration of the conjugated graphene network. These findings confirm that the reduction of GO by ascorbic acid effectively transformed it into rGO by removing oxygen-containing groups.

The TGA curve of pure PVP displayed a sharp and substantial weight loss between 400 and 500 °C, corresponding to the thermal degradation of the polymer backbone. On the other hand, the rGO-PVP composite exhibited a thermal decomposition profile similar to that of rGO. It is worth noting that the PVP content in the composite was only 1% by weight; therefore, no significant difference in thermal stability was observed between the rGO and rGO-PVP curves under the experimental conditions.

3.2. Electrochemical Characterization of the Electrodes

The electrochemical properties of the modified electrodes were investigated using electrochemical impedance spectroscopy (EIS). EIS measurements were performed in an electrolyte solution containing 5 mM [Fe­(CN)₆]^3–/^4– and 0.1 M KCl, with an open-circuit potential set at 0.140 V. The charge transfer resistance (R _ct) of the electrodes was assessed over a frequency range from 100,000 to 0.05 Hz using a 5 mV AC perturbation. In the Nyquist plots, shown in Figure A, the diameter of the semicircle corresponds to the R _ct of the system, and the values obtained were as follows: 4135 Ω for unmodified GCE, 5394 Ω for PVP/GCE, 1236 Ω for rGO/GCE, and 661 Ω for rGO-PVP/GCE. Notably, PVP/GCE exhibited the highest charge transfer resistance, suggesting that insulation by PVP hinders the electron transfer between the redox probes and the electrode surface. rGO/GCE showed a drastically lower charge transfer resistance (nine times lower than that of the unmodified GCE) thanks to the high electrical conductivity of rGO. rGO-PVP/GCE exhibited the lowest R _ct, which can be attributed to PVP stabilizing and preventing rGO aggregation, enhancing the accessibility of redox probes to the electrode surface.

2.

(A) Nyquist plots of the electrodes with different modifications in the presence of 5 mM of [Fe­(CN)₆]^3–/4– in 0.1 M KCl. Inset: Randles equivalent circuit used for data modeling, and (B) cyclic voltammograms of bare GCE, PVP/GCE, rGO/GCE and rGO-PVP/GCE in 5 mM K₃[Fe­(CN)₆] in 0.1 M KCl at a scan rate of 50 mV/s.

The electroactive surface area of the electrodes was measured using cyclic voltammetry with a solution containing 5 mM [Fe­(CN)₆]^3– in 0.1 M KCl. The results, shown in Figure B, were analyzed using the Randles–Sevcik equation at 25 °C

$$i_p=(2.69\times {10}^5)n^{3/2}A{D}^{1/2}C{\nu }^{1/2}$$

where i _p is the peak current for a reversible system; n is the number of the electrons transferred in the redox reaction; A is the electrode area (cm²); D is the diffusion coefficient (cm² s^–1); C is the concentration of redox species (mol cm^–3), and the scan rate (V s^–1). The electroactive surface areas obtained were as follows: 0.0368 cm² for unmodified GCE, 0.0206 cm² for PVP/GCE, 0.0391 cm² for rGO/GCE, and 0.0442 cm² for rGO-PVP/GCE. These results are consistent with the EIS findings and confirm that the incorporation of rGO and PVP improves the electrochemical properties of the electrode, enhancing the electrical conductivity and electroactive surface area.

3.3. Electrochemical Behavior of DA, APAP, and CAF

Cyclic voltammetry was employed to evaluate the performance of the modified electrodes in acetate buffer at pH 4. The first solution, illustrated in Figure A, included DA, APAP, and CAF, while the second solution, shown in Figure B, contained only DA and APAP. Three well-separated oxidation peaks were observed for rGO-PVP/GCE, at 0.43, 0.62, and 1.54 V, respectively. By contrast, rGO/GCE displayed decreased peak currents, and bare GCE exhibited only one oxidation peak for CAF. The combination of rGO and PVP enabled distinct electrochemical responses to DA and APAP. We hypothesize that the presence of PVP aids in stabilizing and dispersing the rGO nanosheets. Furthermore, the phenolic structures in DA and APAD may interact with the imide group of PVP via hydrogen bonding and further boost the peak currents. Consequently, the rGO-PVP/GCE is the best suited design for the simultaneous determination of DA, APAP, and CAF.

3.

(A) CVs of 200 μM DA, 200 μM APAP, and 1 mM CAF in acetate buffer pH 4 at a bare GCE (black), PVP/GCE (red), rGO/GCE (blue), and rGO-PVP/GCE (green), scan rate 50 mV/s, potential range: −0.2 to 1.7 V and (B) CVs of 200 μM DA, 200 μM APAP in acetate buffer pH 4 of the electrodes with different modifications, scan rate 50 mV/s, potential range: −0.2 to 1.0 V.

3.4. Effect of pH

Figure A shows square-wave voltammetry (SWV) curves for a mixture of DA, APAP, and CAF in acetate buffer solutions at different pH values (ranging from 3 to 8). The highest oxidation peak currents were obtained at pH 4 (Figure B) and this pH was chosen for further experiments. The anodic peak potentials (E _pa) of the three compounds varied linearly with the pH (Figure C), with an increase in pH resulting in lower peak potentials, which points to the involvement of protons in the oxidation reactions. The relationships fitted are as follows

$$\mathrm{D}\mathrm{A}{\mathrm{E}}_{\mathrm{p}\mathrm{a}}(\mathrm{V})=-0.0568\mathrm{p}\mathrm{H}+0.6035{\mathrm{R}}^2=0.9909$$

$$\mathrm{A}\mathrm{P}\mathrm{A}\mathrm{P}{\mathrm{E}}_{\mathrm{p}\mathrm{a}}(\mathrm{V})=-0.0403\mathrm{p}\mathrm{H}+0.7248{\mathrm{R}}^2=0.9917$$

$$\mathrm{C}\mathrm{A}\mathrm{F}{\mathrm{E}}_{\mathrm{p}\mathrm{a}}(\mathrm{V})=-0.0075\mathrm{p}\mathrm{H}+1.4725{\mathrm{R}}^2=0.9634$$

4.

(A) Square-wave voltammograms of 200 μM DA, 200 μM APAP, and 1 mM CAF in acetate buffer at different pH values between pH 3 to 8 at rGO-PVP/GCE, (B) plots of I _p vs pH, and (C) plots of E _p vs pH (replicate = 3).

The slopes for DA and APAP were 57 and 40 mV per pH unit, respectively, indicating that the redox reactions of both compounds on the rGO-PVP/GCE surface involved an equal number of protons and electrons, a Nernstian behavior consistent with previous studies. , In particular, the Nernst equation for a reversible electron transfer involving m protons and n electrons is as follows

$$E=E^{0^{\prime }}-\frac{2.303mRT}{nF}\mathrm{p}\mathrm{H}$$

where E ⁰′ is the formal potential of the redox reaction, R is the ideal gas constant, T is the temperature, and F is Faraday constant. For m = n and 25 °C, the potential is expected to decrease 59 mV per pH unit.

For CAF, a slope of −7.5 mV per pH unit suggests a more complex electrochemical process. A previous study proposed a mechanism of CAF oxidation on nickel ferrite/rGO modified electrode involving a two-electron, two-proton oxidation, yielding a substituted uric acid intermediate, followed by a second two-electron, two-proton electro-oxidation, producing the 4,5-diol analogue of uric acid, which then undergoes fragmentation. , Figure S2 summarizes possible electrochemical oxidation reactions of DA, APAP and CAF on rGO-PVP/GCE.

The small slope observed may result from adsorption of oxidation products onto the electrode surface, leading to surface blocking. Regardless, since the highest electrooxidation currents for DA, APAP, and CAF were observed at pH 4, this pH was selected for subsequent experiments.

3.5. Effect of Scan Rate

The effect of scan rate on the electrochemical response of 200 μM DA, 200 μM APAP, and 3 mM CAF at the rGO-PVP/GCE surface was investigated by cyclic voltammetry (CV) in acetate buffer (pH 4.0) over a range of 10–400 mV/s (Figure ). To determine whether the electrochemical oxidation of DA, APAP, and CAF is governed by diffusion or adsorption processes, the relationships between peak current (I _p) and scan rate (ν) as well as ν¹/² were analyzed. Typically, a ln I _p vs lnν plot with a slope near 1.0 indicates adsorption control, while a slope near 0.5 suggests diffusion control. The fitted linear regression equations are shown in Table S1. The ln I _p vs lnν plots for DA, APAP, and CAF yielded slopes of 0.4613, 0.5194, and 0.3992, respectively, with R ² values > 0.99. These results indicate that the redox processes of DA, APAP, and CAF at the rGO-PVP/GCE are limited by diffusion. Moreover, plots of peak potential (E _p) versus scan rate were obtained as shown Figure S3. The peak potential separation (ΔE _p = E _pa–E _pc) for DA and APAP increased with increasing scan rate, suggesting that both analytes undergo quasi-reversible electrochemical processes. In contrast, CAF exhibited an irreversible oxidation behavior, as indicated by the absence of a corresponding reduction peak and the shift of the anodic peak with scan rate.

5.

CVs of (A) 200 μM DA (B) 200 μM APAP (C) 3 mM CAF in acetate buffer pH 4 on rGO-PVP/GCE at different scan rate of 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400 mV/s (D) plot I _p vs ν^1/2 and (E) plot ln I _p vs lnν (replicate = 3).

3.6. Simultaneous Determination of DA, APAP, and CAF

To achieve the simultaneous detection of DA, APAP, and CAF, square-wave voltammetry (SWV) was chosen for its high sensitivity and resolution. rGO-PVP/GCE was used to simultaneously determine DA, APAP, and CAF in two experiments. In a first experiment, the concentration of one species was varied while keeping the other two constant. The resulting SWV data revealed three distinct peaks at potentials of 0.44, 0.63, and 1.49 V (vs Ag/AgCl 3 M KCl), corresponding to the oxidation of DA, APAP, and CAF, respectively. In Figure A, it can be observed that the current response of DA increases with its concentration, while the oxidation currents of APAP and CAF remain relatively constant. Similarly, in Figure B,C, the peak current responses of APAP and CAF increase with their respective concentrations, while the peak currents of the other two fixed compounds remain stable.

6.

Square-wave voltammogram using rGO-PVP/GCE in solution containing; (A) DA (10–200 μM) in the presence of 200 μM APAP and 1 mM CAF; (B) APAP (10–1000 μM) in the presence of 200 μM DA and 1 mM CAF; (C) CAF (40–1000 μM) in the presence of 200 μM APAP and 200 μM DA. Inset shows a calibration plot of the peak currents as a function of analyte concentrations. (D) SWV for different concentrations of DA, APAP and CAF in acetate buffer pH 4.0; (E) Calibration curves of the peak currents as a function of the analyte concentrations at 0.40, 0.53, and 1.43 V for DA, APAP, and CAF, respectively (replicate = 3).

In a second experiment, rGO-PVP/GCE was utilized for the simultaneous determination of DA, APAP, and CAF, with their concentrations varying simultaneously (Figure D). The incremental response to the analytes decreased at high concentrations. Thus, the SWV curves for DA, APAP, and CAF were each fitted to two linear calibration equations at low and high concentration values (Figure E and Table ). The reduced slope observed at high concentrations may be attributed to competitive adsorption of analytes or oxidation products onto the electrode surface.

1. Linear Calibration Curves for DA, APAP and CAF Derived from Figure E.

analyte	concentration range (μM)	linear regression equation	R 2
DA	5–100	Ip = 0.5096 C DA + 2.9067	0.9937
	100–1000	Ip = 0.0543 C DA + 51.6611	0.9768
APAP	5–100	Ip = 0.3687 C APAP – 0.958	0.9945
	100–1000	Ip = 0.0677 C APAP + 36.8513	0.9692
CAF	20–200	Ip = 0.1283 C CAF + 30.462	0.9936
	200–1000	Ip = 0.0398 C CAF + 48.4050	0.9908

Limits of detection (LOD) were calculated using the formula LOD = 3 s/m, where s represents the standard deviation of the blank signal and m is the slope of the linear calibration curve. The calculated LOD values for DA, APAP, and CAF were 0.81, 0.16, and 19.6 μM, respectively. These results were compared with other modified electrodes reported in the literature (Table ). The analytical ranges attained with the proposed sensor for the simultaneous detection of DA, APAP, and CAF are comparable or wider than those reported in previous studies. To our knowledge, no prior study has reported the simultaneous voltammetric determination of DA, APAP, and CAF.

2. Comparison of the Simultaneous Determination of DA, APAP and/or CAF with Different Modified Electrodes Reported in the Literature.

electrode	technique	species	linear range (μM)	detection limit (μM)	ref.
Poly(AHNSA)	SWV	APAP	10–125	0.45
		CAF	51.4–1324	0.87
GCE-M221-Fe3O4	DPV	APAP	50–200	16
		CAF	20–900	23
AG-NA/GCE	DPV	DA	0.5–35	0.33
		APAP	0.05–20	0.031
NiO–CuO/graphene	SWV	DA	0.5–20	0.17
		APAP	4–400	1.33
CeO2–NCs/GCE	DPV	DA	10–400	0.696
		APAP	10–400	0.341
AuNPs-SPCE	DPV	DA	5–200	2.5
		APAP		1.0
carbon black/poly(allylamine HCl)/GCE	LSV	DA	1.0–22	0.55
		APAP	2.4–27	1.3
activation of 3D-Printed	DPASV	APAP	0.4–48	0.44
		CAF	0.9–102	0.58
3D-printed sensor	DPV	APAP	0–115	2.84
		CAF	0–150	2.01
GrRAC	DPV	APAP	0–50	2.01
		CAF	0–50	2.31
rGO-PVP/GCE	SWV	DA	5–100	0.81	this work
			100–1000
		APAP	5–100	0.16
			10–1000
		CAF	20–200	19.6
			200–1000

^aElectrode modification: poly­(AHNSA): poly­(4-amino-3-hydroxynaphthalene sulfonic acid); GCE-M221-Fe₃O₄: cassava starch-Fe₃O₄ nanoparticles modified glassy carbon electrode; AG-NA/GCE: activated graphene/Nafion modified glassy carbon electrode; NiO–CuO/graphene: graphene decorated with nickel and copper oxides; CeO₂–NCs/GCE: cerium oxide nanocubes coated glassy carbon electrode; AuNPs-SPCE: gold nanoparticles modified screen-printed carbon electrode and GrRAC: raw cork (RAC) modified with graphite.

^bElectrochemical technique: MPA = multipulse amperometry; SWV = square-wave voltammetry; LSV = linear sweep voltammetry; DPV: differential-pulse voltammetry and DPASV: differential-pulse anodic stripping voltammetry.

^cAnalyte: DA: dopamine; APAP: acetaminophen and CAF: caffeine.

3.7. Reproducibility, Repeatability and Interference Studies

We investigated the potential impact of various species that may be present in some samples and have oxidation peak potentials similar to those of the analytes. The potential interferents studied included 1 mM glucose, sucrose, citric acid, NH₄ ⁺, Mg²⁺, Ca²⁺, SO₄ ^2–, CO₃ ^2–, and 0.1 mM ascorbic acid in solutions containing 10 μM DA, APAP, and 100 μM CAF. We found that the presence of these species led to small changes in analyte signal (less than 10%), highlighting the robustness of rGO-PVP/GCE for the electrochemical detection of DA, APAP, and CAF.

The reproducibility of the rGO-PVP/GCE design was evaluated by preparing five independent rGO-PVP/GCE electrodes and performing three SWV measurements of the same sample with 10 μM DA, 10 μM APAP, and 100 μM CAF. The relative standard deviation (RSD) values were 3.51% for DA, 3.92% for APAP, and 4.53% for CAF, indicating the rGO-PVP/GCE design is reproducible. The stability of rGO-PVP/GCE was evaluated by measuring the current response for 10 μM DA, 10 μM APAP, and 100 μM CAF over 12 days. After this period, the sensor retained 97.5%, 96.8%, and 95.6% of its initial responses for DA, APAP, and CAF respectively, confirming its high stability.

3.8. Analysis of Real Samples

The rGO-PVP/GCE sensor was used to determine DA, APAP and CAF in an acetaminophen syrup, an energy drink, and a synthetic urine sample using the standard addition method.

A 24 mg/mL commercial acetaminophen syrup was diluted 10-fold with acetate buffer (pH 4.0). 10 μL of the diluted syrup was pipetted into 5 mL of acetate buffer, resulting in a nominal APAP concentration of 31.66 μM. As shown in Table , the relative standard deviation (RSD) obtained with the proposed sensor was +2.56% of the nominal value.

3. Results of DA, APAP and CAF Simultaneous Determination in Acetaminophen Syrup, Energy Drink and Synthetic Urine.

sample	analytes	label amount (μM)	added (μM)	found (μM)	% recovery	% RSD
acetaminophen syrup	APAP	31.66		33.53		2.56
	DA		20	53.6	103.8	1.83
	CAF		20	20.08	100.4	3.47
			100	104.1	104.1	1.76
energy drink	APAP		20	20.87	104.4	2.51
	DA		20	20.14	100.7	1.98
	CAF	85.83		91.97		3.23
			100	195.59	105.2	2.46
synthetic urine	APAP		20	19.38	96.9	3.64
	DA		20	20.67	103.4	1.79
	CAF		100	98.76	98.8	2.60

^a%RSD (Replicate = 3).

In the energy drink case, the CAF concentration indicated on the bottle label was 0.33 mg/mL. After diluting the sample 20-fold, the nominal concentration was 85.83 μM. The CAF concentration obtained using the proposed sensor was 91.97 μM (RSD = +3.23%).

The artificial urine samples were diluted 2 times with 0.1 M acetate buffer (pH 4.0). Then, known amounts of DA, APAP, and CAF were added to the samples. The recoveries determined with the sensor ranged from 96.9% to 105.2% (Table ). From these results, it can be concluded that rGO-PVP/GCE did not suffer from significant interference in the pharmaceutical, beverage and biological samples studied.

4. Conclusion

rGO was synthesized from graphite and GO using ascorbic acid as a green reducing agent. rGO was then integrated with polyvinylpyrrolidone (PVP) to modify a glassy carbon electrode (rGO-PVP/GCE), which exhibited well-resolved oxidation peaks and enhanced current responses in the simultaneous electrochemical determination of DA, APAP, and CAF. The improved performance of the sensor can be attributed to the synergistic interaction between PVP and rGO, which promotes better dispersion of the rGO sheets, increases the electroactive surface area, and enhances the adsorption of phenolic compounds.

Further electrochemical studies confirmed that the oxidation of DA, APAP, and CAF at the rGO-PVP/GCE surface is diffusion controlled, and the sensor operates effectively under a pH of 4. The developed sensor demonstrated excellent selectivity, reproducibility, and stability, with detection limits of 0.81 μM, 0.16 μM, and 19.6 μM for DA, APAP, and CAF, respectively. The sensor analytical ranges for APAP and CAF span the physiological levels found in urine, underscoring its potential biomedical applications. The sensor was successfully applied to real sample analyses, including pharmaceutical syrup, energy drinks, and synthetic urine. The results highlight the potential of rGO-PVP/GCE as a robust and practical system for multianalyte monitoring in clinical diagnostics, pharmaceutical quality control, and beverage analysis.

Glossary

Abbreviations

CAF

Caffeine

CV

cyclic voltammetry

DA

Dopamine

EIS

electrochemical impedance spectroscopy

FE-SEM

field emission scanning electron microscope

FT-IR

Fourier transform infrared spectroscopy

GCE

glassy carbon electrode

GO

graphene oxide

LOD

limit of detection

APAP

acetaminophen

PVP

polyvinylpyrrolidone

rGO

reduced graphene oxide

SWV

square-wave voltammetry

TGA

thermogravimetric analysis

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c02812.

• FE-SEM images of the GCE modified with different materials; proposed electrochemical oxidation mechanisms of DA, APAP and CAF at pH 4; linear regression equations for the correlations peak current (I _p)–square root of scan rate (ν¹/²) and ln I _p–ln ν; and plots of peak potential (E _p) versus scan rate (PDF)

Conceptualization, S.L., V.B. and P.P.; methodology, S.L. and P.P.; validation, S.L., V.B., K.C. and P.P.; investigation, S.L., K.C., J.M. and P.P.; resources, S.L., K.C. and P.P.; data curation, S.L.,V.B. and P.P.; writingoriginal draft preparation, S.L. and P.P.; writingreview and editing, S.L., V.B., K.C., J.M. and P.P.; visualization, S.L., V.B., K.C. and J.M.; supervision, S.L. and P.P.; project administration, S.L. and P.P.; funding acquisition, S.L. and P.P. All authors have approved the final version of the manuscript.

The authors declare no competing financial interest.