Reductant-free Synthesis of Gold Nanoparticles on Citric Acid/Carbon Nanotube Matrix for the Simultaneous Detection of Ascorbic Acid, Dopamine, and Uric Acid
Article information
Abstract
Ascorbic acid (AA), dopamine (DA), and uric acid (UA) are biologically important molecules whose concentrations are closely related to several diseases such as Parkinson’s disease, Alzheimer’s disease, and gout. Therefore, real-time and accurate monitoring of these analytes is crucial for early diagnosis and treatment. Although various analytical methods such as Raman scattering, chromatography, fluorescence, and electrochemical techniques have been developed, electrochemical sensing is particularly attractive due to its high sensitivity, fast response, and cost-effectiveness, making it suitable for portable biosensing platforms. However, electrochemical detection of AA, DA, and UA faces challenges due to their overlapping oxidation potential, which hinders selective analysis. To overcome this, electrode surface modification using nanomaterials—such as metal nanoparticles, graphene, and carbon nanotubes—has been proposed to enhance sensitivity and enable peak separation. In this study, we fabricated a modified glassy carbon electrode (GCE) by drop-casting citric acid-functionalized multi-walled carbon nanotubes (CA-MWCNTs) and reducing gold ions (Au3+) in situ using citric acid to form gold nanoparticles (AuNPs). This reductant-free synthesis enables facile control over nanoparticle formation simply by adjusting the gold ion concentration and incubation time, without the need for toxic chemical reductants or high-temperature conditions required in conventional methods. The resulting AuNPs/CA-MWCNT/GCE was characterized by SEM and EDS, and its electrochemical properties were investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The modified electrode exhibited distinct oxidation peaks for AA, DA, and UA at −0.088 V, 0.148 V, and 0.276 V, respectively, with detection limits of 300 μM, 1.5 μM, and 6.0 μM. The electrode also showed pH-dependent oxidation behavior, high selectivity against common interferents, and excellent long-term electrochemical stability. This simple fabrication approach is suitable for large-scale production and can be extended to flexible substrates, offering potential for wearable electrochemical sensors.
INTRODUCTION
Dopamine (DA), ascorbic acid (AA), and uric acid (UA) play important physiological functions in the human body, and changes in their concentrations are closely linked to various diseases. DA, as a neurotransmitter, is involved in motor control, emotion, and cognitive functions, and when affected by neurodegenerative and psychiatric disorders. The altered concentration of dopamine can serve as a biomarker for these diseases such as Parkinson’s disease, Alzheimer’s disease, and schizophrenia [1–3]. AA possesses strong antioxidant properties, plays a critical role in protecting cells from oxidative stress and supporting immune and neurological functions, and its deficiency can lead to disorders such as scurvy and immune suppression [4–6]. UA is the final product of purine metabolism and contributes to oxidative defense in the body; however, excessive accumulation is associated with gout, kidney stones, and cardiovascular diseases [7–9]. In particular, recent studies have demonstrated that quantitative analysis of changes in DA, AA, and UA concentrations, and their effects on neuroinflammatory responses, oxidative stress, and metabolic balance, provides important insights into physiological processes [10]. Such analyses can serve as valuable indicators for early disease diagnosis, prediction of disease progression, and the development of personalized treatment strategies [11,12]. Therefore, real-time and precise quantitative analysis of AA, DA, and UA is essential for clinical diagnosis, disease monitoring, and treatment response evaluation, and securing the detection accuracy and portability of these substances along with the advancement of biosensor technology is considered a core task for implementing future precision medicine.
To date, various analytical techniques, including Raman scattering, chromatography, fluorescence, and electrochemical methods, have been employed to detect AA, DA, and UA, each offering distinct advantages in specific applications. Raman spectroscopy provides non-destructive structural analysis with high spectral resolution, allowing selective detection of biomolecules such as proteins and disease markers. Particularly, surface-enhanced Raman spectroscopy (SERS) further amplifies Raman signals on nanoparticle surfaces, enabling ultra-sensitive detection for precision diagnostic applications including single-cell and tissue-level analyses [13–15]. Chromatography (HPLC, GC, etc.) can separate analytes from the complex media, and quantitative analysis with trace level of analytes, making it extensively used in pharmaceutical, environmental, and food analysis [16,17]. Fluorescence analysis offers high sensitivity and is widely applied in biomedical research for detecting biomarkers and intracellular substances. Recently, it has also evolved into a powerful tool for simultaneous quantitative and qualitative analysis [18–20]. However, these methods generally have limitations such as expensive equipment, complex sample pretreatment, long analysis times, constraints exist for performing real-time and field-based analysis.
In comparison, electrochemical analysis methods directly apply samples to electrodes and measure current or voltage changes, offering advantages such as high sensitivity, fast response, low equipment cost, miniaturization and portability potential, and simple preparation [21,22]. Due to these advantages, they have recently been attracting attention as core technologies in the advantages of smart healthcare, point-of-care diagnostic devices, and wearable devices [23–25]. However, despite these advantages, electrochemical methods have the problem that AA, DA, and UA have similar oxidation potentials, causing electrochemical peak overlap, which becomes a factor that hinders the accuracy of selective detection [26,27].
To overcome these limitations, modifying electrode surfaces with nanomaterials to simultaneously improve effective surface area and electrical conductivity is necessary. In electrochemical measurements, electron transfer rate and mass transfer rate are key factors determining sensor performance. Electron transfer depends on the electrical conductivity and surface characteristics of electrode materials, while mass transfer is governed by diffusion, convection, and migration. Therefore, various nano-based electrode modification technologies using metal nanoparticles, graphene oxide (rGO), carbon nanotubes (CNTs), and MXene have been developed. These materials provide benefits such as increased electron transfer efficiency, expanded effective surface area, and improved detection resolution [28–32].
In this study, we employed a citric acid (CA)-functionalized multi-walled carbon nanotubes (CA-MWCNT) and gold nanoparticles (AuNPs) as electrode functionalization materials. The electrode functionalization was performed by drop-casting method on the surface of a glassy carbon electrode (GCE), enabling the simultaneous and individual detection of AA, DA, and UA. The MWCNT was functionalized by CA to enhance dispersity and increase the binding site for gold cations. Furthermore, the CA functional groups can be a reducing agent for AuNPs. In this work, the term “reductant-free” indicates that no external or dedicated chemical reducing agents (such as NaBH4 or hydrazine) were added during AuNP formation; the citric acid present in the CA-MWCNT matrix acts simultaneously as a mild reductant, surface functionalizer, and stabilizer without the need for additional reducing species. AuNPs act as electron transfer catalysts for electrochemical reactions, improving the sensor’s sensitivity. CA-MWCNT promotes π–π interactions with the aromatic rings of AA, DA, and UA, yielding distinct oxidation potentials through variations in electron transfer rate. This electrode design can be said to be an effective strategy not only for surface area expansion but also for securing selectivity and signal amplification in electrochemical reactions.
Schematic diagrams for the fabrication of AuNPs/CA-MWCNT/GCE for simultaneous detection of AA, DA and UA.
The surface characteristics of the AuNPs/CA-MWCNT/GCE electrode were confirmed with SEM, EDS, FTIR, and water contact angle (WCA) analyses, and evaluated the electrochemically effective surface area through CV. The differential pulse voltammetry (DPV) was obtained to determine the analytical performance of AuNPs/CA-MWCNT/GCE for simultaneous and individual detection of AA, DA, and UA. Additionally, the electrochemical reaction mechanism, selectivity over the potential sensing interferences, and stability were performed to evaluate sensing performance of the AuNPs/CA-MWCNT/GCE.
This research presents a comprehensive approach to developing advanced electrochemical biosensors for simultaneous detection of important biological markers, combining the benefits of AuNPs and functionalized CNTs to overcome traditional limitations in electrochemical sensing while enabling future applications in wearable and point-of-care diagnostic devices.
EXPERIMENTAL
Reagents and Materials
Citric acid (CA), cysteamine, phenylalanine, potassium hexacyanoferrate(II) trihydrate (K4Fe(CN)6·3H2O), tyrosine, gold(III) chloride trihydrate (HAuCl4·3H2O), L-ascorbic acid (AA), dried powder of phosphate buffered saline (PBS), potassium hexacyanoferrate(III) (K3Fe(CN)6), uric acid (UA), dopamine hydrochloride (DA), and urea were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Potassium nitrate (KNO3) was obtained from Samchun Pure Chemical Co., LTD (Pyeongtaek, Gyeonggi-do, Korea). Hydrochloric acid (35.0~37.0%) (HCl) was obtained from Duksan Pure Chemicals Co., LTD (Ansan, Gyeonggi-do, Korea). Sodium hydroxide (beads, above 97%) (NaOH) was obtained from Daejung Chemicals & Metals Co., LTD (Siheung, Gyeonggi-do, Korea). All chemicals used were of analytical grades and aqueous solutions were prepared with deionized (DI) water. DI water was prepared using water purification systems (specific resistivity >18.2 MΩ·cm, PURE ROUP 50, Purewater, Namyang-ju, Gyeonggi-do, Korea).
Instrumentation
CV and differential pulse voltammetry (DPV) experiments were performed using a CHI 620A electrochemical workstation (CH Instruments, Inc., Austin, USA). A conventional three-electrode cell was used at room temperature, including glassy carbon electrode (GCE, BAS Inc., Tokyo, Japan) as working electrode, Pt wire (BAS Inc., Tokyo, Japan) as counter electrode, and RE-1B Ag/AgCl electrode (BAS Inc., Tokyo, Japan) as reference electrode. Scanning electron microscopy (SEM) images and energy dispersive spectroscopy (EDS) spectrum were taken on a S-4800 (Hitachi, Tokyo, Japan, operated at 15 kV using P-1 Glassy Carbon plate (BAS Inc., Japan). Fourier transform infrared (FT-IR) spectra were performed Perkin Elmer Spectrometer 100 (Waltham, Massachusetts, USA). Contact angle measurements were performed on a PHOENIX-MINI (SEO Co, Ltd., Suwon, Gyeonggi-do, Korea) and P-1 Glassy Carbon plate (BAS Inc., Japan) was used to substrate for contact angle images. Ultrasonic dispersion was carried out using Q125 ultrasonic processor with 1/8" ultrasonic probe (QSonica, Newtown, USA).
Fabrication of AuNPs/CA-MWCNT/GC electrode
Pristine MWCNTs were ultrasonically dispersed for 30 min in an aqueous solution of 0.5 mg/mL CA using Q125 ultrasonic processor with 1/8" ultrasonic probe (QSonica, Newtown, USA). A glassy carbon electrode (GCE; ø 3 mm) was sequentially polished with 1.0, 0.3, and 0.05 μm alumina slurries, then sonicated in deionized water for 5 min and rinsed thoroughly. Next, 30 μL of the CA-MWCNT suspension was dropcast onto the polished GCE and dried under an infrared lamp (IR 250 RH IR2, 250 W, Philips, Netherlands). for 50 min. The modified electrode was rinsed with deionized water, dried under N2, then immersed for 2 h in 2 mL of 1 mM HAuCl4 in 0.1 M KNO3 (Fig. 1). Finally, the AuNPs/CA-MWCNT/GCE was rinsed with deionized water and dried under N2.
(A) UV–Vis spectra of 2 mL 1 mM HAuCl4 in 0.1 M KNO 3 after addition of 30 μL 1 mg/mL CA-MWCNT, were recorded with different incubation time of 0, 1, 5, 10, 30, 60, 120, 150, and 180 min after CA-MWCNT addition. (B, C) UV–Vis spectra (200 to 1100 nm) of 2 mL 1 mM HAuCl4 (0.1 M KNO3) were recorded with different incubation time of 0, 1, 5, 10, 30, 60, 120, 150, and 180 min with immersing of CA-MWCNT/GCE. All measurements were performed in triplicate (n = 3), and the error bars represent the standard deviations.
To optimize the immersion time for AuNP formation, UV–Vis spectra were recorded under two conditions: i) 2 mL of 1 mM HAuCl4 in 0.1 M KNO3 solution was placed in a quartz cuvette and scanned from 200 to 1100 nm. Then, 30 μL of well-dispersed 1 mg/mL CA-MWCNT was added to the solution, and spectra were immediately recorded with different incubation time of 0, 1, 5, 10, 30, 60, 120, 150, and 180 min. ii) 2 mL of 1 mM HAuCl4 in 0.1 M KNO3 solution was again scanned from 200 to 1100 nm. Subsequently, a CA-MWCNT/GCE was immersed into the solution, and spectra were recorded with different incubation time of 0, 1, 5, 10, 30, 60, 120, 150, and 180 min.
RESULTS AND DISCUSSION
Preparation and characterization of AuNPs/CA-MWCNT/GCE
The reductant-free synthesis of AuNPs by using CA-MWCNT was exhibited with UV-Vis spectrometry. As shown in Fig. 1, the absorbance of the Au3+ ligand-to-metal charge transfer (LMCT) band at 320 nm decreased with incubation time, allowing 120 min to be selected as the optimized incubation time. Furthermore, the increasing absorption band around 600 nm was observed that indicating the localized surface plasmon resonance of the formed AuNPs. Therefore, an incubation time was selected as 120 min for AuNPs formation. These results indicate that our CA-MWCNT based reductant-free synthesis of AuNPs is effectively achieved.
Due to the incompatibility of the modified GCE with scanning electron microscopy (SEM), a glassy carbon plate (GCP) was obtained for surface characterization of GCE when modified with the same method as described in Fig. 1. The surface characteristics of the modified electrode were then exhibited by using SEM and energy dispersive spectroscopy (EDS) elemental analysis. As shown in Fig. 2, the SEM images illustrate the differences in surface characteristics between the bare GCP (Fig. 2A) and the AuNPs/CA-MWCNT/GCP (Fig. 2B). The SEM image of the bare GCP exhibits a smooth carbon plate surface, whereas the AuNPs/CA-MWCNT/GCP showed an overall rough texture with entangled rod-like structures and sphere-shaped particles. The size of the AuNPs was measured to be approximately 190 nm ± 20 nm. The EDS analysis spectrum of AuNPs/CA-MWCNT/GCE confirms the presence of carbon, oxygen, and gold. The carbon signal originates from the glassy carbon plate and carbon nanotubes, while the oxygen signal is attributed to the hydroxyl (-OH) groups introduced from CA. The gold signal is derived from the AuNPs on the electrode surface (Fig. 2B). Furthermore, EDS mapping images of AuNPs/CA-MWCNT/GCE was demonstrating the distribution of carbon and oxygen throughout the electrode surface. Notably, the regions containing white nanoparticles correspond to the presence of gold, further confirming that these nanoparticles are indeed gold nanoparticles (Fig. S1). The resulting surface characteristics confirm the successful fabrication of the AuNPs/CA-MWCNT/GCE by the presented method.
SEM images of (A) bare GCE and (B) AuNPs/CA-MWCNT/GCE. (C) EDS analysis spectrum of AuNPs/CA-MWCNT/GCE
To further confirmation of electrode surface characteristics, WCA was introduced which can evaluate the hydrophilicity of substrate. As shown in Fig. 3, WCA for each step of electrode (GCE, MWCNT/GCE, CA-MWCNT/GCE, and AuNPs/CA-MWCNT/GCE) was measured. The water contact angle of the bare GCE was 77.9°, indicating a relatively hydrophobic surface. The MWCNT-coated GCE exhibited an increased contact angle of 93.9°, reflecting its intrinsic hydrophobicity. by nature of MWCNT. CA-MWCNT/GCE showed a dramatical decrease in WCA to 44.2° due to added –COOH and –OH groups. Finally, the AuNPs/CA-MWCNT/GCE displayed the lowest contact angle of 27.6°, demonstrating the greatest enhancement in surface hydrophilicity. In summary, the developed AuNPs/CA-MWCNT/GCE exhibits surface characteristics favorable for application in water-based sensing environments.
WCA images of (A) bare GCE, (B) MWCNT/GCE, (C) CA-MWCNT/GCE, and (D) AuNPs/CA-MWCNT/GCE.
Electrochemical property of AuNPs/CA-MWCNT/GCE
CV measurements were performed to examine the electrochemical properties of AuNPs/CA-MWCNT/GCE. Fig. 4A presents the cyclic voltammograms of bare GCE, MWCNT/GCE, CA-MWCNT/GCE, and AuNPs/CA-MWCNT/GCE, recorded in 0.1 M KNO3 containing 5 mM [Fe(CN)6]3– at a scan rate of 50 mV/s. The redox peak currents increased in the order of bare GCE < MWCNT/GCE < CA-MWCNT/GCE < AuNPs/CA-MWCNT/GCE. The high redox peak currents indicate that this electrode has an effective electrochemical surface area and electrocatalytic activity. To determine the effective electrochemical surface area, CV measurements were performed with different scan rates of 10, 50, 100, and 500 mV/s (Fig. 4B), and the effective electrochemical surface areas were calculated with the Randles-Sevcik equation [33,34]; the estimated surface areas for bare GCE, MWCNT/GCE, CA-MWCNT/GCE, and AuNPs/CA-MWCNT/GCE were 0.038, 0.11, 0.094, and 0.14 cm2, respectively. Nanomaterials such as MWCNTs and AuNPs significantly enhance the effective electrochemical surface area of the modified GCE, indicating that these nanomaterials were successfully incorporated onto the electrode surface. These results demonstrate that the modification of GCE with CA-MWCNT and AuNPs improves its electrochemical catalytic activity and enlarges the effective surface area, which synergistically facilitates efficient electron transfer.
(A) CV curves recorded on bare GCE, MWCNT/GCE, CA-MWCNT/GCE, and AuNPs/CA-MWCNT/GCE in 0.1 M KNO3 containing 5 mM ferricyanide- at a scan rate of 50 mV/s. and (B) Relationship between peak current and the square root of scan rate for oxidation on bare GCE, MWCNT/GCE, CA-MWCNT/GCE, and AuNPs/CA-MWCNT/GCE in 0.1 M KNO3 containing 5 mM ferricyanide. All measurements were performed in triplicate (n = 3), and the error bars represent the standard deviations.
The electrochemical properties obtained from ferricyanide CV and EIS measurements are summarized in Table 1, including effective electrochemical surface area, current density, peak-to-peak separation (ΔEp), and charge-transfer resistance (Rct). These probe-based metrics indicate that both MWCNT/GCE and AuNPs/CA-MWCNT/GCE provide fast and efficient electron-transfer rate, reflecting the inherently high conductivity of the CNT network and the expanded effective surface area introduced by AuNPs. The AuNPs/CA-MWCNT/GCE, in particular, combines the advantages of CNT-mediated charge transport with the additional catalytic sites and enhanced surface accessibility offered by the Au nanoparticles, resulting in a robust electrochemical interface suitable for sensitive redox reactions.
Electrochemical properties of the electrodes obtained from ferricyanide CV and EIS measurements.
While ferricyanide serves as a general indicator of electron-transfer capability, the sensing performance for target biomolecules is governed not only by charge-transport kinetics but also by analyte-specific interactions and catalytic activity at the electrode surface. The distinctive contributions of AuNPs and CA functionalization become more evident in the oxidation behavior of AA, DA, and UA, which is discussed in detail in Section 3.3.
Detection performance of AuNPs/CA-MWCNT/GCE for AA, DA, and UA
DPV measurements were performed to evaluate the analytical performance of the AuNPs/CA-MWCNT/GCE for both individual and simultaneous electrochemical detection of AA, DA, and UA (Fig. 5). The bare GCE exhibited a single broad oxidation peak around 0.21 V, indicating poor resolution among AA, DA, and UA. In contrast, the AuNPs/CA-MWCNT/GCE displayed three well-defined and distinct oxidation peaks at – 0.088, 0.148, and 0.276 V, corresponding to AA, DA, and UA, respectively. Additional DPV measurements using MWCNT/GCE and CA-MWCNT/GCE also produced separated oxidation peaks for AA, DA, and UA, and the corresponding curves are provided in the Supplementary Information (Fig. S2). However, the peak currents obtained with these intermediate electrodes were noticeably lower than those of the AuNPs/CA-MWCNT/GCE, indicating that the presence of Au nanoparticles considerably enhances the signal intensity and practical sensitivity required for reliable simultaneous detection.
(A) DPV curves of bare GCE and AuNPs/CA-MWCNT/GCE in 0.1 M PBS containing 1 mM AA, 50 μM DA and 50 μM UA. (B) DPV curves of AuNPs/CA-MWCNT/GCE at various concentrations of AA (100, 200, 300, 500, 700, 900 and 1000 μM), DA (3, 5, 10, 20, 50, 70 and 100 μM) and UA (10, 30, 50, 70, 100, 150 and 200 μM) in 0.1 M PBS (pH 7.4).
This clear separation of oxidation peaks can be attributed to the superior electrochemical catalytic properties of the AuNPs/CA-MWCNT composite, which enhances electron transfer kinetics and minimizes peak overlap. To further assess the sensor’s simultaneous detection capability, DPV responses were recorded under increasing concentrations of AA (100–1000 μM), DA (3–100 μM), and UA (10–200 μM) in 0.1 M PBS (pH 7.4). Fig. 5B illustrates that the oxidation peak currents of all three analytes increased linearly with their respective concentrations, confirming the electrode’s excellent sensitivity and selectivity toward each species. Taken together, these results indicate that the AuNPs/CA-MWCNT/GCE was successfully fabricated as a reliable electrochemical sensor for the simultaneous determination of AA, DA, and UA.
DPV measurements were further conducted to evaluate the analytical performance of the AuNPs/CA-MWCNT/GCE in terms of linear range and limit of detection (LOD) for AA, DA, and UA (Fig. 6). Fig. 6A, 6C, and 6E show the DPV responses obtained in 0.1 M PBS (pH 7.4) by varying the concentration of each target analyte—AA (100–1000 μM), DA (1–80 μM), and UA (10–200 μM)—while maintaining the other two at constant levels (1 mM AA, 50 μM DA, and 50 μM UA). The oxidation peak currents for AA, DA, and UA increased proportionally with their respective concentrations, confirming a sensitive and stable electrochemical response. Calibration plots derived from the DPV data (Fig. 6B, 6D, and 6F) were used to determine the linear range and LOD of each analyte.
DPV curves of AuNPs/CA-MWCNT/GCE in 0.1 M PBS (pH 7.4) containing different concentrations of (A) AA (from inner curve to outer curve: 0, 100, 200, 300, 500, 700, 900 and 1000 μM), (C) DA (from inner curve to outer curve: 0, 1, 5, 10, 20, 40, 60 and 80 μM) and (E) UA (from inner curve to outer curve: 0, 10, 30, 50, 70, 100, 150 and 200 μM). (B, D, F) Corresponding calibration curves. Each value corresponds to replicated experiments (n = 3).
The differences in slope and linearity among the three calibration curves arise from the distinct molecular interactions and electron-transfer behaviors of AA, DA, and UA on the CNT/Au composite surface. DA, with its catechol structure, exhibits stronger π–π stacking and adsorption onto carbon-based nanomaterials, leading to higher sensitivity, whereas UA shows weaker surface affinity and partially diffusion-controlled kinetics, resulting in a smaller slope and mild curvature at higher concentrations. These analyte-dependent variations have been widely reported for CNT-based electrochemical sensors [35,36].
The LOD values for AA, DA, and UA were estimated to be 300, 1.5, and 6.0 μM, respectively (calculated as LOD = 3.3 × S/N), and the corresponding analytical parameters are summarized in Table 2. These findings demonstrate that the AuNPs/CA-MWCNT/GCE provides reliable quantitative detection performance toward AA, DA, and UA, highlighting its strong potential for electrochemical sensing applications.
Summary of the analytical performance of AuNPs/CA-MWCNT/GCE for the electrochemical determination of AA, DA, and UA
Table 3 summarizes the analytical performance of the AuNPs/CA-MWCNT/GCE compared with recently reported metal nanoparticles and/or carbon-nanomaterial–based sensors. The sensitivity of our electrode is comparable to these systems, while requiring fewer fabrication steps owing to its simple, reductant-free construction. This demonstrates the practical competitiveness of the proposed sensor.
Comparison of various electrode materials for the electrochemical detection of AA, DA, and UA.
pH effect
AA, DA, and UA undergo pH-dependent electrochemical redox reactions, with protons involved in the electrochemical oxidation process. To examine the pH effect on these analytes using AuNPs/CA-MWCNT/GCE, DPV was performed at various pH values (4.0–10.0), and the oxidation peak potentials (Epa) were compared. As shown in Fig. 7A, the Epa values of AA, DA, and UA shifted progressively toward more negative potential as the pH increased. The theoretical slope for pH-dependent electrochemical reactions is 59 mV/pH [40], and the experimentally determined slopes for AA, DA, and UA were 33, 67, and 53 mV/pH, respectively, which are close to theoretical value. The variations in slope among AA, DA, and UA arise from differences in their proton-coupled electron-transfer kinetics and surface interactions with the CNT/Au composite, which cause each analyte to exhibit slightly different pH sensitivities. These results indicate that the oxidation of AA, DA, and UA entails an equal number of protons and electrons, as confirmed by Fig. 7B, which shows that the process specifically involves two protons and two electrons. These findings confirm the pH-dependent electrochemical behavior of AA, DA, and UA and provide valuable insights into their oxidation mechanisms at AuNPs/CA-MWCNT/GCE.
Plots of anodic peak potential vs. pH value for the electrochemical mechanism study of 1 mM AA, 50 μM DA and 50 μM UA in 0.1 M PBS buffer solution. (B) Possible oxidation mechanisms for AA, DA and UA, respectively. All measurements were performed in triplicate (n = 3), and the error bars represent the standard deviations.
Selectivity and stability test
Selectivity is a crucial factor in biosensors, particularly in minimizing interference from other biological materials. To confirm the selectivity of AuNPs/CA-MWCNT/GCE and assess whether interferents affect the current response, chronoamperometry measurements were performed using AuNPs/CA-MWCNT/GCE. During the measurement, 100 μM AA, 20 μM DA, and 10 μM UA were sequentially introduced every 50 seconds, along with 10 μM of citric acid, cysteamine, tyrosine, phenylalanine, and urea as potential interferents and the applied potentials were -0.088, 0.148, and 0.276 V for AA, DA, and UA, respectively. As shown in Fig. 8, the amperometric responses of AuNPs/CA-MWCNT/GCE remained stable despite the addition of interferents, indicating no significant current changes. These results suggest that AuNPs/CA-MWCNT/GCE have good selectivity for AA, DA, and UA unaffected by potential interferents.
Amperometric responses of AuNPs/CA-MWCNT/GCE upon successive addition of (A) 100 μM AA, (B) 20 μM DA, (C) 10 μM UA, and other chemicals of 10 μM to 0.1 M PBS buffer (pH 7.4). The applied potentials for AA, DA and UA are −0.088, 0.148, and 0.276 V, respec vely.
Ferricyanide was selected as the redox probe for the long-term stability test because its reversible and well-defined electron-transfer behavior provides a reliable measure of intrinsic electrode stability without interference from analyte-specific effects. Fig. 9 presents the long-term electrochemical stability of the electrode, assessed CV tests conducted over 250 cycles at a scan rate of 50 mV/s. The CV curves showed negligible changes with no apparent deviations observed throughout the cycles (Fig. 9A). The percentage change in peak current as a function of the number of cycles is presented in Fig. 9B. The current retained 99.6% of its initial value even after 250 cycles of the ferricyanide redox process, the current remained highly stable without significant degradation (Fig. 9B). These results demonstrate that AuNPs/CA-MWCNT/GCE exhibits excellent electrochemical stability, maintaining consistent performance even after repeated redox cycles.
Stability test of AuNPs/CA-MWCNT/GCE in 0.1 M KNO3 containing 5 mM ferricyanide up to 250 cycles at a scan rate 50 mV/s.
CONCLUSIONS
In this study, AuNPs/CA-MWCNT/GCE electrode was developed for the simultaneous and individual electrochemical detection of DA, AA, and UA. CA-MWCNT provided enhanced dispersibility, abundant surface oxygen groups, and - interaction sites, while in situ grown AuNPs served as highly efficient electron-transfer mediators and electrocatalysts. Comprehensive physicochemical characterization (SEM, EDS, FT-IR, WCA) confirmed successful fabrication of AuNPs/CA-MWCNT/GCE. Electrochemical evaluation by CV demonstrated an enlarged effective electrode area, and differential pulse voltammetry achieved clear separation of oxidation peaks at –0.088 (AA), 0.148 (DA), and 0.276 V (UA) (vs. Ag/AgCl reference electrode) with detection limits of 300, 1.5, and 6.0 μM, respectively. Furthermore, pH-dependent studies, interference assays, and long-term stability tests validated the sensor’s selectivity, reproducibility, and durability. The simple drop-casting and dipping fabrication protocol is amenable to large-scale production and adaptable to flexible substrates, opening routes to wearable or point-of-care biosensors. Overall, the integration of CA-MWCNT and AuNPs offers a powerful strategy for overcoming peak-overlap challenges in electrochemical sensing, paving the way for real-time, portable, and precise monitoring of critical biomolecules in complex biological environments.
Notes
ACKNOWLEDGMENTS
This work was supported by a research grant from Seoul Women’s University (2023-0008).
CONFLICTS OF INTEREST
The authors declare no conflicts of interest.