Poly(Allura Red AC)-Modified Glassy Carbon Electrode: Electropolymerization, Characterization, and Application in Uric Acid Detection

Abstract

In this study, the electropolymerization, electrochemical characterization, and analytical applicability of a poly­(Allura Red AC)-modified glassy carbon electrode were systematically investigated. Electropolymerization parameters, including monomer concentration, buffer type, pH, and ionic strength, were optimized to obtain a stable and electroactive poly­(Allura Red AC) film. The resulting polymer film was characterized by cyclic voltammetry, electrochemical impedance spectroscopy, surface coverage analysis, electroactive surface area determination, and pH-dependent electrochemical studies. The modified electrode exhibited enhanced electrocatalytic activity toward the oxidation of uric acid, enabling sensitive voltammetric determination. Under optimized conditions, the sensor showed a linear range of 0.2–40 μM and low detection limits of 7.64 nM and 6.47 nM by cyclic voltammetry and differential pulse voltammetry, respectively. The developed methods were successfully applied to protein-free human serum samples, providing accurate recoveries and no prominent interfering peaks within the uric acid oxidation potential window. This work provides a comprehensive understanding of the formation and electrochemical properties of poly­(Allura Red AC) films while demonstrating their applicability as an electrocatalytic sensing interface for sensitive uric acid determination in biological samples.

1. Introduction

Electroactive polymers have emerged as a highly attractive class of organic materials for electrochemical sensor design due to their superior electrical conductivity, large surface area, facile functionalization, biocompatibility, and cost-effective preparation. Among the various synthesis methods, electropolymerization of redox-active monomers offers a particularly versatile and controllable approach that allows the in situ formation of redox-active films with strong adhesion, tunable thickness, and good electrochemical stability. Such films have been widely applied in sensing platforms targeting biomolecules, pharmaceutical compounds, and environmental pollutants. ,

Azo dyes, defined by their −NN– azo linkage and extended π-conjugation, represent a prominent subgroup of electroactive compounds that are frequently used in the fabrication of conductive polymer films. These dyes are widely used in the textile, food, cosmetic, and pharmaceutical industries due to their intense coloration, high stability, and economic synthesis. , A synthetic dye belonging to this group, Allura Red AC (AR) (disodium 6-hydroxy-5-[(2-methoxy-5-methyl-4-sulfonatophenyl)­diazenyl]­naphthalene-2-sulfonate), is approved for use in food and pharmaceutical products. Its electron-rich aromatic structure and multiple sulfonic acid groups enhance both its redox activity and solubility in aqueous media. These characteristics make AR a promising monomer for electropolymerization. The resulting poly­(AR) films offer enhanced electron transfer kinetics and strong surface affinity for analyte adsorption, making them effective materials for voltammetric sensing applications. ,

Electrochemical sensors based on chemically modified electrodes continue to attract significant interest in analytical science due to their simplicity, high sensitivity, and cost-effectiveness. , Recent studies have shown that functionalized electrode materials and polymer-based or hybrid sensing interfaces can enhance electron transfer, electrocatalytic activity, sensitivity, and selectivity in the electrochemical detection of biologically relevant analytes. − Electropolymerization of functional monomers onto electrode surfaces remains one of the most effective strategies for sensor development, enabling the formation of stable and conductive interfaces with excellent selectivity and reproducibility. ,

Uric acid (UA) is a key biomarker associated with purine metabolism disorders such as gout, hyperuricemia, and renal dysfunction. In healthy individuals, the normal blood UA level is between 0.15 and 0.42 mM; however, impaired metabolism or excretion may lead to pathological accumulation. A below-normal level of UA in the blood serum is called hypouricemia and is often considered a sign of a medical condition such as heavy metal poisoning and some types of cancer. Therefore, accurate and sensitive detection of UA is clinically relevant. Conventional analytical techniques, including liquid chromatography-mass spectrometry, fluorescence, chemiluminescence, enzymatic assays, and electrochemical methods, have been developed to monitor UA levels in biological fluids. − Among these, voltammetric sensors have emerged as a preferred alternative due to their high sensitivity, low detection limits, minimal sample preparation requirements, and suitability for miniaturization. Recent studies have demonstrated that modified electrodes can improve the electrocatalytic oxidation, sensitivity, selectivity, and simultaneous determination of UA in the presence of dopamine, with successful applications in human serum samples. − In this study, the electropolymerization of AR on a glassy carbon electrode (GCE) was systematically investigated using cyclic voltammetry (CV). The resulting poly­(AR)-modified electrode, which can be readily prepared through a simple one-step procedure, was characterized and subsequently employed for the voltammetric determination of UA in both standard solutions and spiked serum samples. The proposed platform provided a rapid and sensitive analytical response under the investigated conditions. Overall, this work provides new insights into the electrochemical formation, electrochemical characteristics, and electrocatalytic performance of poly­(AR) films, while demonstrating their applicability as an efficient electrocatalytic sensing interface for UA determination in biological samples.

2. Materials and Methods

2.1. Reagents and Materials

AR (dye content: 80%, molecular weight: 496.42 g mol^–1), l-ascorbic acid (AA), and dopamine hydrochloride (DA) were purchased from Sigma-Aldrich. UA was supplied by Acros Organics. Human serum (AB plasma, sterile-filtered, USA origin) was also purchased from Sigma-Aldrich.

Buffer solutions were prepared using analytical-grade reagents. Phosphate buffer solutions (PB, 0.2 M) at pH 6.0, 7.0, and 8.0 were prepared using NaH₂PO₄·2H₂O and Na₂HPO₄ (Sigma-Aldrich). Acetate buffer solutions (AcB, 0.2 M) at pH 3.5, 4.5, and 5.5 were prepared using CH₃COOH (99.8%, d = 1.049 g mL^–1, Merck). Britton–Robinson buffer solutions (BRB, 0.04 M) were prepared over a pH range of 3.0 to 10.0 by mixing equimolar solutions of CH₃COOH, H₃PO₄ (85.0%, d = 1.685 g mL^–1, Merck), and H₃BO₃ (99.5%, Sigma-Aldrich), followed by pH adjustment using 5.0 M NaOH. To investigate the effect of ionic strength, 0.1 M solutions of KCl, KNO₃, and Na₂SO₄ (Sigma-Aldrich) were employed.

All solutions were prepared with ultrapure water, and all chemicals were used without further purification.

2.2. Instrumentation

Electrochemical measurements were performed using a Metrohm Autolab PGSTAT128N potentiostat/galvanostat (Metrohm-Autolab, Netherlands) equipped with NOVA 1.11 software. A conventional three-electrode system was employed, consisting of a poly­(AR)/GCE as the working electrode, a platinum wire as the auxiliary electrode, and an Ag/AgCl (3.0 M KCl) electrode as the reference electrode. The pH was measured using an HI2211 pH meter (Hanna Instruments). Ultrapure water from a Sartorius Arium Pro UV system (Germany) was used for solution preparation.

2.3. Preparation of Poly­(AR)/GCEs

The bare GCE was polished with 0.05 μm alumina slurry on a polishing pad, followed by ultrasonic cleaning in ultrapure water for 10 min. To eliminate background current, the electrode was preconditioned by CV scanning in buffer solution between −0.5 and +1.3 V at a scan rate of 100 mV s^–1 for 10 cycles.

Electropolymerization of AR on the GCE surface was carried out by CV within a potential window of −0.5 to +1.3 V at a scan rate of 100 mV s^–1 using 0.5 mM AR solutions prepared in PB (pH 6.0–8.0), AcB (pH 3.5–5.5), and BRB (pH 3.0–10.0). Different electrolytes (KCl, KNO₃, and Na₂SO₄) at a concentration of 0.1 M were tested to evaluate the effect of ionic strength on polymer film formation. In addition, the effect of AR monomer concentration was investigated by varying it from 0.1 to 1.0 mM to optimize electropolymerization conditions. After polymerization, the obtained poly­(AR)/GCEs were rinsed with ultrapure water and dried at room temperature for 24 h before use.

2.4. Electrochemical Determination of UA

The electrochemical determination of UA was performed using CV and differential pulse voltammetry (DPV) at the poly­(AR)/GCE. CV measurements were performed using a step potential of 5 mV and a scan rate of 100 mV s^–1. DPV measurements were performed using a step potential of 5 mV, a modulation amplitude of 50 mV, a modulation time of 50 ms, an interval time of 0.50 s, and a scan rate of 10 mV s^–1. All measurements were carried out at room temperature in freshly prepared BRB (pH 5.0) containing appropriate concentrations of UA.

Calibration curves were constructed by plotting the peak current (I _p) versus UA concentration, and regression analysis was performed to determine analytical parameters, including linear range, limit of detection (LOD), limit of quantification (LOQ), and precision. LOD and LOQ were calculated using the expressions 3s/m and 10s/m, respectively, where s is the standard deviation of the peak currents obtained from three replicate measurements at the lowest calibration concentration, and m is the slope of the corresponding calibration curve. , Method precision was evaluated using five replicate measurements for both intra-day and inter-day analyses, and the results were expressed as percent relative standard deviation (%RSD).

2.5. Analysis of Spiked Human Serum Samples

The applicability of the developed electrode was evaluated through recovery experiments using spiked human serum samples. Serum samples were stored at sub-zero temperatures and protected from light prior to analysis. UA was spiked into a defined serum aliquot to obtain a stock solution containing 0.1 mM UA, followed by the addition of acetonitrile to induce protein precipitation. The final volume was adjusted to 2.0 mL using the original serum matrix. A volume ratio of 1.5:1 (acetonitrile:serum, v/v) was applied to ensure efficient protein removal. The mixture was vortexed for 30 s and centrifuged at 5000 rpm for 10 min. The resulting supernatant was collected and appropriately diluted with buffer solution to obtain working solutions for electrochemical analysis.

2.6. Interference Studies

The selectivity of the method toward UA was evaluated in the presence of common electroactive interferents, including DA and AA. In the first set of experiments, the DA concentration was fixed at 1 μM while the UA concentration was varied from 6 to 80 μM. In the second set, the AA concentration was maintained at 140 μM, and UA was varied over the same concentration range. Additional experiments were conducted by varying DA (1–10 μM) and AA (80–180 μM) concentrations while keeping the UA concentration constant at 20 μM. DPV measurements were employed to monitor changes in I _p and assess potential interference effects.

3. Results and Discussion

3.1. Electrochemical Polymerization of AR on GCE

Electropolymerization of AR onto the GCE was carried out by CV in the potential range of −0.5 V to +1.3 V (vs. Ag/AgCl) at a scan rate of 100 mV s^–1 for 50 cycles. Figure presents the cyclic voltammograms recorded during electropolymerization of 0.4 mM AR in pH 5.0 BRB. A prominent irreversible oxidation peak was observed at +0.82 V. This response is consistent with previous experimental and theoretical studies of AR electrooxidation on glassy carbon electrodes, which identified the azo moiety as the principal electroactive center and proposed the formation of an azo-centered radical cation following electron transfer. The gradual decrease in peak intensity with increasing cycle number indicates continuous monomer consumption and progressive growth of a polymeric film on the electrode surface.

1.

Cyclic voltammograms of 0.4 mM AR in pH 5.0 BRB. Scan rate 100 mV s^–1.

Characteristic redox features corresponding to different stages of electropolymerization were observed. Such azo-centered radical species may subsequently participate in coupling and surface-film-forming reactions during repeated potential cycling, thereby contributing to the growth of the poly­(AR) film. A lower-potential redox couple initially observed around +0.18 V is tentatively attributed to the redox response of electroactive monomer-derived intermediate species formed during the early stages of polymerization. The increasing peak-to-peak separation (ΔE _p) and decreasing I _p values indicate progressively hindered electron transfer and reduced electroactive accessibility during polymer formation. The gradual decrease in its peak current with successive cycles reflects progressive monomer consumption and conversion into polymeric structures. In contrast, the redox couple centered around +0.11 V progressively intensified with successive cycles, consistent with the continuous growth and increasing electroactivity of the poly­(AR) film. The gradual shift of the anodic peak toward more positive potentials may reflect changes in the local electrochemical environment and accessibility of redox-active sites during film growth.

Following polymerization, the appearance of a pale red layer on the electrode surface provides visual evidence of successful coating with the poly­(AR) film.

The effects of buffer type and pH on the electropolymerization of 0.5 mM AR were investigated using BRB, PB, and AcB solutions over a pH range of 3.0–10.0. No monomer oxidation peaks were observed at pH 8.0, 9.0, and 10.0 in BRB or at pH 8.0 in PB, indicating that AR is electrochemically inactive under these conditions. Cyclic voltammograms recorded during AR electropolymerization in BRB (pH 3.0–7.0), AcB (pH 3.5–5.5), and PB (pH 6.0 and 7.0) revealed that the fastest and most controlled polymer growth, along with the most uniform poly­(AR) film formation, was achieved in pH 5.0 BRB.

The influence of monomer concentration on electropolymerization was subsequently examined, and 0.4 mM AR was found to yield well-defined polymer formation and the highest I _p of poly­(AR). The effect of common anions (Cl^–, NO₃ ^–, and SO₄ ^2–) on poly­(AR) formation was then evaluated by performing electropolymerization in pH 5.0 BRB containing 0.4 mM AR and 0.1 M KCl, KNO₃, or Na₂SO₄. Analysis of the cyclic voltammograms showed that the polymer peak currents decreased in the presence of these ions; therefore, subsequent polymerization experiments were conducted in pH 5.0 BRB without additional supporting electrolytes.

The surface coverage (Γ, mol cm^–2) of the poly­(AR) films was calculated from the charge (Q) corresponding to the anodic peak using eq :

$$\mathrm{\Gamma }=Q/nFA$$ 1

where n is the number of electrons transferred per monomer unit (assumed to be 2 based on literature), F is the Faraday constant (96485 C mol^–1), and A is the geometric surface area of the GCE (7.069 × 10^–2 cm²). ,, Table summarizes the charge and surface coverage values calculated from cyclic voltammograms of poly­(AR) films electropolymerized at different pH values using 0.4 mM AR and a scan rate of 100 mV s^–1. Among the investigated conditions, poly­(AR)₃ synthesized in pH 5.0 BRB exhibited the highest surface coverage, reaching 11.43 nmol cm^–2.

1. Charges Corresponding to Polymer Oxidation and Surface Concentration of Poly­(AR) Films.

Polymer	Buffer used for electropolymerization	Q/μC cm–2	Γ/nmol cm–2
Poly(AR)1	BRB, pH 3.0	3.25	4.05
Poly(AR)2	BRB, pH 4.0	3.54	4.39
Poly(AR)3	BRB, pH 5.0	9.20	11.43
Poly(AR)4	BRB, pH 6.0	2.51	3.12
Poly(AR)5	BRB, pH 7.0	1.54	1.92

SEM imaging was performed to examine the surface morphology of the bare GCE and the poly­(AR)₃/GCE (Figure S1). The SEM images obtained at 35,000× magnification showed that the bare GCE exhibited a relatively uniform surface morphology, whereas the poly­(AR)₃/GCE displayed a distinctly altered and more heterogeneous surface with irregular morphological features. These observations are consistent with modification of the GCE surface by the electropolymerized poly­(AR) layer.

3.2. Electrochemical Characterization of Poly­(AR) Films

The electrochemical behavior of poly­(AR)/GCEs was examined using CV in BRB solutions over a pH range of 5.0–9.0. Buffer solutions with pH values below 5.0 or above 9.0 were excluded to ensure the stability of the polymer film. Among the films listed in Table , the poly­(AR)₃ film exhibited the most symmetrical redox peaks, the highest I _p values, and the strongest linear correlation between I _p and scan rate. Therefore, this electrode was selected for subsequent studies.

Figure shows the cyclic voltammograms of poly­(AR)₃/GCE recorded at scan rates ranging from 5 to 200 mV s^–1. A well-defined redox peak pair corresponding to the redox transitions of the poly­(AR) film was observed. With increasing scan rate, both anodic and cathodic I _p values increased linearly, while the peak potential (E _p) shifted toward more positive values for the anodic peak and more negative values for the cathodic peak. To evaluate the reversibility of the redox process, the ΔE _p was examined as a function of scan rate. At a scan rate of 5 mV s^–1, ΔE _p was approximately 45 mV and increased to 102 mV at 200 mV s^–1. This increase in ΔE _p with scan rate, together with an anodic-to-cathodic I _p ratio of approximately 0.30 at 100 mV s^–1, supported the quasi-reversible nature of the electron-transfer process.

2.

Cyclic voltammograms of poly­(AR)₃/GCE in pH 5.0 BRB at scan rates of 5, 10, 15, 25, 50, 75, 100, 150, and 200 mV s^–1.

The linear relationship between I _p and scan rate confirms that the redox process is predominantly governed by adsorption-controlled kinetics. The corresponding slope values for the anodic and cathodic processes, along with other relevant parameters, are summarized in Table . In addition, the slope obtained for the oxidation process was higher than that for the reduction process, indicating that the expulsion of the positive counterion from the polymer film occurs more rapidly than its diffusion into the film. This behavior is consistent with previous reports on azo dye-based conductive polymer films. ,

2. Linear Regression Results of I _p vs. Scan Rate Plots of Poly­(AR)₃ Film in pH 5.0 BRB.

	Oxidation process	Reduction process
Slope, μA s V–1	20.15	18.75
Intercept, μA	0.18	0.13
Correlation coefficient	0.991	0.998

The effect of pH on the electrochemical response of poly­(AR)₃ was also examined. As shown in Figure , plots of redox E _p versus pH exhibited linear relationships with slopes of −59.3 mV pH^–1 for the oxidation peaks and −52.8 mV pH^–1 for the reduction peaks. These values are close to the theoretical Nernstian slope (−59 mV pH^–1) expected for a redox process involving an equal number of protons and electrons. In addition, Figure demonstrates that both anodic and cathodic I _p values of the polymer film decrease linearly with increasing pH.

3.

Dependence of I _p and E _p of poly­(AR)₃ on the pH of BRB. Scan rate 100 mV s^–1. Squares indicate oxidation, triangles indicate reduction; filled symbols refer to I _p, unfilled symbols refer to E _p.

Electrochemical impedance spectroscopy (EIS) is a powerful technique for evaluating the success of electrode surface modification. The interfacial properties of the bare GCE and the poly­(AR)₃/GCE were investigated by EIS in a 0.1 M KCl solution containing 1.5 mM [Fe­(CN)₆]^3–/4–. As shown in Figure , the Nyquist plots exhibit semicircular features with different diameters in the high-frequency region, indicating changes in the charge-transfer resistance (R_ct) at the electrode interfaces. The R_ct value represents the resistance to electron transfer between the electrode surface and the redox probe. Accordingly, the R_ct value was 1264.45 Ω for the bare GCE, whereas it significantly decreased to 128.57 Ω after modification with poly­(AR)₃. This pronounced decrease in R_ct confirms the enhanced electrical conductivity provided by the poly­(AR)₃ film. The improved conductivity is attributed to the electroactive nature of the poly­(AR)₃ layer, which facilitates electron transport and reduces interfacial resistance between the electrode and electrolyte. To further elucidate the interfacial processes, the EIS data were fitted using Randles equivalent circuit (inset of Figure ), where R_s, C_dl, CPE, and Z_w represent the solution resistance, double-layer capacitance, constant phase element, and Warburg impedance, respectively.

4.

Nyquist plots of bare GCE and poly­(AR)₃/GCE recorded in 0.1 M KCl containing 1.5 mM [Fe­(CN)₆]^3–/4–. Symbols: experimental data; solid lines: equivalent-circuit fits. Frequency range: 0.1–10⁵ Hz. Applied potential: +0.25 V. Amplitude: 0.01 V.

The increase in the charge-transfer coefficient after modification (from 0.8930 to 0.9252) suggests the formation of a more homogeneous and uniform electrode surface, which likely facilitates more efficient charge transport. In addition, the pronounced decrease in Z_w from 5.69 × 10^–4 to 7.91 × 10^–7 S s^–1/2 indicates that ion diffusion at the electrode surface is significantly enhanced by poly­(AR)₃ film. The decrease in Y₀ (from 1.66 × 10^–5 to 2.98 × 10^–6 S s^a) and C_dl values implies the formation of a more compact and controlled interfacial structure on the modified electrode surface. Overall, these results demonstrate that electrode modification with poly­(AR) is successful, leading to improved charge-transfer characteristics and supporting its potential for electrochemical sensing applications.

3.3. Determination of Electroactive Surface Area

The electroactive surface areas of the bare GCE and the poly­(AR)₃/GCE were evaluated by CV using a 1.5 mM [Fe­(CN)₆]^3–/4– solution prepared in 0.1 M KCl. Voltammograms were recorded at scan rates ranging from 10 to 150 mV s^–1, and the corresponding anodic I _p values were used to calculate the electroactive surface areas according to eq :

$$I_{\mathrm{pa}}=2.69\times {10}^5{n}^{3/2}AC{D}^{1/2}{v}^{1/2}$$ 2

where I _pa is the anodic I _p (A), n is the number of electrons involved in the redox process (n = 1), A is the electroactive surface area (cm²), C is the concentration of the redox probe (mol cm^–3), D is the diffusion coefficient (7.6 × 10^–6 cm² s^–1), and v is the scan rate (V s^–1). The linear relationship observed between I _p and v ^1/2 confirms a diffusion-controlled redox process and validates the use of this equation.

Compared to the bare GCE, poly­(AR)₃/GCE exhibited significantly higher I _p values, indicating enhanced electron-transfer kinetics and an increased electroactive surface area resulting from the electropolymerized film. Based on the slopes of the linear I _p versus v ^1/2 plots, the electroactive surface areas were calculated to be 0.062 cm² for the unmodified GCE and 0.104 cm² for the poly­(AR)₃/GCE. These results demonstrate that modification of the GCE with the poly­(AR)₃ film increases the electroactive surface area by approximately 1.68-fold.

3.4. Electroanalytical Investigation of UA at Poly­(AR)₃/GCE

The electrochemical performance of poly­(AR)₃/GCE toward UA oxidation was evaluated using both CV and DPV. As shown in Figure , the modified electrode exhibited a significantly enhanced oxidation current response compared to the bare GCE. The anodic peak current of 40 μM UA was 2.39 μA at the bare GCE and 4.89 μA at the poly­(AR)₃/GCE. This approximately two-fold increase in I _p confirms the effective electrocatalytic activity of the polymer film toward UA oxidation. In addition, poly­(AR)₃/GCE displayed improved baseline stability and a higher signal-to-noise ratio.

5.

Differential pulse voltammograms of 40 μM UA in pH 5.0 BRB obtained with (a) bare GCE and (b) poly­(AR)₃/GCE.

3.5. Effect of pH and Scan Rate

The effect of pH on the anodic response of UA was investigated using BRB solutions over a pH range of 5.0–9.0 (Figure ). In the CV measurements (Figure A), a single irreversible anodic peak was observed in the absence of a corresponding cathodic peak, confirming the irreversible nature of the oxidation process. The highest I _p value was obtained at pH 5.0, corresponding to oxidation potentials of approximately +490 mV for CV and +440 mV for DPV. With increasing pH, the anodic E _p shifted toward more negative values, accompanied by a gradual decrease in I _p, consistent with a proton-coupled oxidation mechanism. Accordingly, pH 5.0 was selected as the optimum medium for subsequent experiments due to its superior peak shape, higher current response, and improved electrochemical performance.

6.

(A) Cyclic and (B) differential pulse voltammograms of 10 μM UA in BRB at pH (a) 5.0, (b) 6.0, (c) 7.0, (d) 8.0, and (e) 9.0 on poly­(AR)₃/GCE.

A linear relationship between E _p and pH was observed in the DPV measurements, with a slope of −61.4 mV pH^–1, which is in close agreement with the theoretical Nernstian value of −59 mV pH^–1. This finding suggests that the oxidation of UA involves an equal number of protons and electrons. Based on previous literature, UA oxidation is proposed to proceed through an initial irreversible two-electron/two-proton transfer, resulting in the formation of an oxidized intermediate. The poly­(AR)₃ film facilitates UA oxidation by increasing the electroactive surface area and promoting interfacial electron transfer, as supported by the CV and EIS results. The proposed electrocatalytic oxidation mechanism of UA at the poly­(AR)₃/GCE is presented in Scheme .

1. Proposed Electrocatalytic Oxidation Mechanism of UA at the Poly­(AR)₃/GCE.

The effect of scan rate on the electrochemical behavior of UA at poly­(AR)₃/GCE was investigated by CV over a scan rate of 25–300 mV s^–1 in pH 5.0 BRB. As the scan rate increased, the E _p of 10 μM UA shifted toward more positive values, which is characteristic of an irreversible electrochemical process. A linear relationship was obtained in the log I _p versus log v plot, described by eq :

$$\log I_{\mathrm{p}}(\mu \mathrm{A})=0.740\log v-1.846(r=0.997)$$ 3

In addition, the I _p–v plot also exhibited linearity within the investigated scan rate range, as expressed by eq :

$$I_{\mathrm{p}}(\mu \mathrm{A})=0.03v+0.115(r=0.993)$$ 4

The slope value of 0.740 obtained from the log I _p–log v plot suggests that the UA oxidation process at the poly­(AR)₃/GCE is influenced by both diffusion and adsorption. The linear I _p–v relationship further supports the presence of adsorptive contributions. Therefore, the electrochemical oxidation of UA at the modified electrode is considered to involve mixed diffusion- and adsorption-controlled behavior. Since consistent and reproducible preconcentration conditions could not be established for differential pulse stripping voltammetry, subsequent quantitative measurements were performed using direct CV and DPV without a deposition step.

3.6. Determination of UA with Poly­(AR)/GCE

Quantitative analysis of UA was carried out using poly­(AR)₃/GCE by both CV and DPV. As shown in Figure , a well-defined and proportional increase in the I _p was observed with increasing UA concentration over the range of 0.2–40 μM. The corresponding calibration data are summarized in Table .

7.

(A) Anodic segments of cyclic voltammograms and (B) differential pulse voltammograms obtained in pH 5.0 BRB containing UA at various concentrations: (a) 0.4 μM, (b) 4 μM, (c) 10 μM, (d) 20 μM, and (e) 40 μM. Dashed lines: Blank. Insets: Calibration plots of I _p vs. UA concentration.

3. Analytical Parameters Obtained for the Determination of UA in pH 5.0 BRB Using Poly­(AR)₃/GCE.

	CV	DPV
E p [mV]	493	437
Linear range [μM]	0.2–40	0.2–40
Slope [μA μM–1]	4.88 × 10–2 ± 9.55 × 10–4	9.14 × 10–2 ± 2.06 × 10–3
Intercept [μA]	–1.15 × 10–2 ± 1.30 × 10–2	4.37 × 10–2 ± 2.80 × 10–2
Correlation coefficient	0.996	0.995
LOD [nM]	7.64	6.47
LOQ [nM]	23.2	19.6
Repeatability of E p [%RSD]	0.56	0.50
Repeatability of I p [%RSD]	0.75	0.80
Intermediate precision of E p [%RSD]	0.92	1.12
Intermediate precision of I p [%RSD]	1.22	1.35

^aUA concentration: 20 μM, number of experiments: 5.

The LODs were determined to be 7.64 nM for CV and 6.47 nM for DPV, which are among the lowest values reported for similar electrochemical sensors (Table ). Repeatability was evaluated using five replicate measurements under intra-day conditions, while intermediate precision was assessed under inter-day conditions, demonstrating good analytical precision with %RSD values below 1.35% for both I _p and E _p. In addition, electrode-to-electrode reproducibility was evaluated using five independently prepared poly­(AR)₃/GCEs. The electrodes were tested by DPV using 20 μM UA under identical experimental conditions, and the oxidation peak currents and peak potentials exhibited %RSD values of 2.83% and 0.52%, respectively, indicating good reproducibility of the electrode fabrication procedure.

4. Comparison of Analytical Parameters Obtained Using Different Electrochemical Sensors for UA Determination.

Electrode	Method	Linear range [μM]	LOD [μM]	Reference
GR-SWCNT–Ce-Cu-Tween 20/GCE	DPV	0.08–100	6.3 × 10–3
MWCNTs/poly(AHNSA)/GCE	SWV	1.0–100	0.024
MOF-235/GCE	DPV	10–900	3.46
CS@-Fe2O3@Ag/GCE	DPV	0.5–8	0.042
FN/rGO/GCE	DPV	4.0–21.5	1.3
MWCNT-Zn(II)TAPc/GCE	CV	0.05–1.2	0.066
CuNPs/TAT/GCE	DPV	0.03–1100	2.9 × 10–3
g-C3N4/GO/GCE	DPV	0.009–90	2.1 × 10–3
Poly(AR)3/GCE	CV	0.2–40	7.64 × 10–3	This work
	DPV	0.2–40	6.47 × 10–3

^aCopper and cerium bimetallic nanoparticles and carbon nanomaterials of graphene and single-walled carbon nanotubes in the presence of Tween 20/glassy carbon electrode.

^bMultiwalled carbon nanotubes-poly­(4-amino-3-hydroxynaphthalene sulfonic acid)/glassy carbon electrode.

^cMetal-organic framework-235/glassy carbon electrode.

^dCore-shell carbon sphere/Fe₂O₃@Ag/glassy carbon electrode.

^eNickel ferrite/reduced graphene oxide/glassy carbon electrode.

^fMultiwalled carbon nanotubes-tetraamino zinc­(II) metallophthalocyanine/glassy carbon electrode.

^gCopper nanoparticles–tartrazine nanocomposite/glassy carbon electrode.

^hGraphitic-like carbon nitride/graphene oxide composite nanosheets/glassy carbon electrode.

Given the clinical importance of UA and the need for its rapid and cost-effective determination, various modified electrodes have been developed to construct selective, sensitive, and low-detection-limit electrochemical sensors. In this context, the performance of the poly­(AR)₃/GCE sensor developed in the present study was compared with previously reported systems (Table ). As summarized in Table , poly­(AR)₃/GCE exhibits promising analytical performance compared with previously reported systems, highlighting its potential as a viable alternative for UA detection. The sensor provides a wide linear response range along with remarkably low limits of detection, reflecting its enhanced sensing capability. Although some reported modified electrodes exhibit slightly lower detection limits, ,, their fabrication typically involves multi-step and labor-intensive modification procedures. In contrast, the poly­(AR)₃/GCE sensor achieves competitive sensitivity and high precision through a rapid and straightforward electropolymerization process, highlighting its suitability as a practical platform for routine UA analysis.

3.7. Determination of UA in Spiked Serum Samples

The applicability of the developed poly­(AR)₃/GCE for the quantification of UA in biological matrices was evaluated using protein-free spiked human serum samples prepared in pH 5.0 BRB. CV and DPV measurements performed with these samples produced well-defined oxidation peaks within the UA oxidation potential window, without any prominent interfering peaks, supporting the applicability of the sensor in the investigated serum matrix. The corresponding recovery results are summarized in Table .

5. Recovery Results for UA in Serum Samples Prepared in pH 5.0 BRB Determined Using Poly­(AR)₃/GCE.

	CV	DPV
Added concentration of UA [μM]	0.2000	0.2000
Obtained concentration of UA [μM]	0.1988	0.2005
Average recovered [%]	99.39	100.24
%RSD of recovery	1.41	1.14
Bias%	–0.60	0.25

3.8. Interference Studies

Selectivity is a critical parameter in the electrochemical determination of UA, particularly in complex biological matrices where electroactive species such as DA and AA may coexist. Accordingly, the selectivity of the poly­(AR)₃/GCE toward UA was evaluated through interference studies using DPV.

In the first set of experiments, the DA concentration was fixed at 1 μM while the UA concentration was varied between 6 and 80 μM. In the second set, the AA concentration was maintained at 140 μM and the UA concentration was varied over the same range. In both cases, the oxidation current of UA increased linearly with increasing UA concentration, indicating that the UA response could be quantitatively monitored in the presence of DA and AA. The following eqs and show the linearity obtained in the first and second cases:

$$I_{\mathrm{p}}(\mathrm{A})=0.0256C(\mathrm{M})-4.0\times {10}^{-8}(r=0.996)$$ 5

$$I_{\mathrm{p}}(\mathrm{A})=0.0351C(\mathrm{M})-1.0\times {10}^{-7}(r=0.991)$$ 6

Additional interference studies were performed by varying the concentrations of DA (1–10 μM) and AA (80–180 μM) while keeping the UA concentration constant at 20 μM. As shown in Figure , in the presence of DA, the UA peak current remained nearly unchanged up to 6 μM DA, with a maximum variation of approximately 2.5%. At higher DA concentrations (8–10 μM), a more pronounced increase in the UA peak current was observed; however, the UA oxidation peak remained clearly distinguishable. In the presence of AA, the UA peak current decreased from 0.393 μA at 80 μM AA to 0.325 μA at 100 μM AA. Thereafter, the UA response remained relatively stable over the AA concentration range of 100–180 μM, with the peak current varying between 0.320 and 0.336 μA. The corresponding calibration equations are given in eqs and :

$$I_{\mathrm{p}}(\mathrm{A})=0.1053C(\mathrm{M})-5.0\times {10}^{-8}(r=0.996)$$ 7

$$I_{\mathrm{p}}(\mathrm{A})=0.0031C(\mathrm{M})-9.0\times {10}^{-8}(r=0.994)$$ 8

8.

Differential pulse voltammograms of (A) DA and (B) AA at various concentrations in the presence of 20 μM UA. Inset: Plots of I _p vs. DA or AA concentration.

Overall, the interference studies demonstrate that the UA oxidation peak remains distinguishable in the presence of DA and AA, although some variation in the UA peak current was observed depending on the interferent concentration. These results support the selectivity of the poly­(AR)₃/GCE toward UA under the investigated conditions.

3.9. Stability of the Modified Electrode

The stability of the poly­(AR)₃/GCE was evaluated by storing the sensor at room temperature under dry conditions and periodically recording its response using DPV in pH 5.0 BRB containing 8 μM UA. After 30 days of storage, the sensor retained approximately 91% of its initial current response, indicating limited signal degradation (Figure S2). This good stability can be attributed to the strong adhesion of the electropolymerized film to the electrode surface, as well as the chemical robustness of the azo dye backbone. These characteristics highlight the potential of the poly­(AR)₃/GCE as a reliable platform for routine electrochemical sensing applications.

4. Conclusions

In this study, a novel electrochemical sensor was successfully fabricated via electropolymerization of AR onto the surface of a GCE. Optimization of polymerization parameters, including pH, monomer concentration, and ionic strength, led to the formation of a stable and electroactive polymer layer with high surface coverage and strong adhesion to the electrode surface.

The poly­(AR)₃/GCE exhibited good electrocatalytic activity toward the oxidation of UA, enabling its sensitive and selective determination using both CV and DPV. The developed sensor showed a wide linear response range, remarkably low detection limits, and good repeatability, intermediate precision, electrode-to-electrode reproducibility, and selectivity. In addition, the sensor maintained stable performance over extended storage periods, underscoring its suitability for practical analytical applications.

Importantly, the proposed method was successfully applied to the determination of UA in spiked human serum samples, achieving high accuracy, minimal matrix interference, and reliable performance in a biological matrix. Overall, these results demonstrate that the poly­(AR)₃/GCE is a promising platform for the sensitive determination of UA and has potential for further development toward practical biological applications. Furthermore, this study highlights the broader potential of azo dye-derived conductive polymers as cost-effective and high-performance materials for electrochemical sensor development. Future research may focus on extending this strategy to other clinically and environmentally relevant analytes, as well as exploring composite or nanomaterial-supported poly­(AR) films to further enhance sensitivity and multifunctionality.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c09469.

• SEM images of the bare GCE and poly­(AR)₃/GCE, and differential pulse voltammetric data for the 30-day stability evaluation of the poly­(AR)₃/GCE (PDF)

The authors declare no competing financial interest.