Thiol-mediated electrochemical sensor for the breast cancer biomarker CA 15-3

Cancer antigen 15-3 (CA 15-3), a circulating fragment of the MUC1 glycoprotein, is widely used for monitoring breast cancer progression. In this study, we report a label-free, receptor-free electrochemical detection strategy based on adsorption-mediated modulation of gold surface oxidation. Pencil graphite electrodes (PGEs) were modified with electrochemically deposited gold nanoparticles (AuNPs) using a reproducible multi-electrode fabrication system. The nanostructured AuNP surface exhibited enhanced electroactive area and improved electron-transfer properties, as confirmed by ferri/ferrocyanide characterization. Analytical detection was performed in phosphate-buffered saline by monitoring the intrinsic gold oxidation peak. Following incubation with CA 15-3, a concentration-dependent decrease in the gold oxidation current was observed, attributed to adsorption-induced surface passivation of electrochemically active gold sites. The platform demonstrated a linear response across the tested concentration range with a calculated limit of detection of 1.2 × 10 −4 U/mL. Measurements in spiked plasma samples confirmed the feasibility of detecting CA 15-3 in complex matrices under controlled conditions. This work establishes a chemically driven, adsorption-based electrochemical sensing strategy that exploits gold surface chemistry for label-free biomarker detection.

Cancer is a leading cause of death worldwide, with around 10 million deaths in 2020, a number expected to reach 16 million by 2040 (Ferrlay et al., 2021). One of the major challenges contributing to this high mortality is the late-stage diagnosis of many cancers, which limits treatment options and lowers survival rates. Breast cancer is the most frequently diagnosed cancer in women and a major cause of cancer-related deaths, responsible for 685,000 deaths and 2.26 million new cases in 2020 alone. It accounts for 24.5% of all female cancers. In 2018, breast cancer led to 630,000 deaths, with 2.09 million new diagnoses (Iweala et al., 2024). In China, incidence rose by 17.6% and mortality by 15.6%, presenting growing public health challenges (Iweala et al., 2024). Despite advances in detection and treatment, traditional screening methods like mammography and biopsy have significant drawbacks. Mammography, while effective in older women, is less accurate in younger women or those with dense breast tissue, often leading to false positives. Biopsies, though definitive, are invasive, time-consuming, and can produce false negatives. Early detection is critical for survival, especially in low- and middle-income countries, where limited access to diagnostic tools leads to more late-stage diagnoses. Risk factors for breast cancer include age, genetic mutations (e.g., BRCA1/2), family history, and lifestyle factors like diet, obesity, and alcohol use. A promising avenue for early detection is through biomarkers, such as proteins, RNA, metabolites, and circulating tumor cells (CTCs). Biomarkers enable earlier, more precise diagnosis and disease monitoring, offering a less invasive and potentially more accurate alternative to traditional methods. Biomarkers are molecules that signal the presence or progression of disease. In breast cancer, they include proteins, RNA, and circulating tumor cells (CTCs), which undergo changes as cancer develops (Jain and Joshi, 2023). Since noticeable symptoms often appear only after tumors have significantly progressed, early detection in asymptomatic individuals is essential. This requires non-invasive or minimally invasive methods using small samples of fluids like blood or urine, with high sensitivity and specificity to detect cancer-related biomarkers at an early stage. One widely studied breast cancer biomarkers is CA 15-3. CA 15-3, also known as Mucin 1, is a glycoprotein linked to breast cancer. Normal serum levels are below 30 U/mL, but 30%–50% of breast cancer patients show levels above 100 U/mL (Li et al., 2023). CA 15-3 is useful for assessing disease severity, monitoring recurrence, and tracking metastasis. It is also elevated in cancers such as ovarian, lung, and colorectal (Dolsch et al., 2017). MUC1 is a high–molecular weight transmembrane glycoprotein characterized by a variable number tandem repeat (VNTR) region, extensive O-linked glycosylation, multiple cysteine residues and aberrant glycosylation patterns in cancer (Kargar et al., 2022). CA 15-3 corresponds to circulating fragments of the extracellular domain of MUC1 released into the bloodstream. In malignant conditions, MUC1 undergoes altered glycosylation and conformational modifications that may expose peptide regions normally shielded in healthy cells. Structural analyses indicate that cysteine residues participate in disulfide bond formation; however, under certain environmental or interfacial conditions, partial unfolding or surface interactions can expose sulfur-containing groups. Consequently, the extracellular domain of MUC1 possesses structural features capable of interacting with metallic interfaces, particularly nanostructured gold, and the cancer-associated glycoform alterations further modulate its adsorption behavior (Kargar et al., 2022; Nath and Mukherjee, 2014; Kufe, 2009). Although CA 15-3 is valuable for monitoring cancer, traditional detection methods like ELISA and ECLIA are limited by low sensitivity, long processing times, and labeling requirements. These challenges highlight the need for more advanced, rapid, and sensitive diagnostic technologies. Immunochemical methods, which rely on specific antibody-antigen interactions, are highly effective for detecting these biomarkers. Immunosensors, a subset of these techniques, enhance detection speed and accuracy in clinical diagnostics (Patil et al., 2022; Li et al., 2010; Oliveira et al., 2024a; Oliveira et al., 2024a; Martins et al., 2022; Marques et al., 2018; Farzin and Naghib, 2024; Qin et al., 2019; Ghanbari et al., 2018; Amani et al., 2018). As breast cancer rates continue to rise globally, incorporating biomarkers like CA 15-3 into routine screening is critical. These markers support early diagnosis, guide treatment decisions, and enable personalized care. Continued advances in biomarker technology, combined with increased public awareness and screening initiatives, are essential to improving breast cancer outcomes and reducing mortality (Ribeiro et al., 2021). To enable sensitive detection of breast cancer biomarkers like CA 15-3, sensor material strategies must evolve alongside device architecture. Biosensors represent a promising alternative for breast cancer biomarker detection due to their ease of use, cost-effectiveness, and rapid response time; features that align well with clinical and patient needs (Panicker et al., 2024; Kummari et al., 2023; Rao Bommi et al., 2023). Various biosensing techniques have been investigated for cancer detection, including colorimetric, electrochemical, piezoelectric, and optical methods. Among these, electrochemical approaches have emerged as particularly advantageous owing to their high sensitivity, selectivity, low cost, user-friendliness, and quick response times. In these approaches, researchers have employed diverse biological recognition elements to detect target biomarkers.

Gold nanostructures are widely employed in biosensors because of their high electrical conductivity, excellent biocompatibility, strong chemical stability, large surface-to-volume ratio, and ease of surface functionalization (Gul et al., 2025; Katz et al., 2004). In electrochemical systems, gold nanoparticles significantly enhance analytical performance by increasing the electroactive surface area, improving charge-transfer kinetics, providing a high density of immobilization sites, and enabling signal amplification (Guo and Wang, 2007; Dykman and Khlebtsov, 2012; Liu et al., 2023; Lien et al., 2020). Gold-based biosensors have been successfully applied for the detection of proteins, nucleic acids, small molecules, and cancer biomarkers, as the nanostructured interface promotes efficient electron transfer at the electrode surface while supporting adsorption or covalent attachment of biomolecules. Importantly, electrodes modified with gold nanoparticles often exhibit lower limits of detection due to enhanced interfacial sensitivity and increased binding site density (Gul et al., 2025; Guo and Wang, 2007; Dykman and Khlebtsov, 2012; Ge et al., 2012; Momeni et al., 2022; Katz et al., 2004); in receptor-free strategies, the nanostructured gold serves both as a signal transduction enhancer and as a chemically interactive platform. Gold–thiol (Au–S) chemisorption is one of the most extensively studied surface chemistries in nanoscience and electrochemistry, arising from the strong affinity between gold atoms and sulfur-containing functional groups, particularly thiols (–SH). Upon adsorption, thiols typically undergo dissociative chemisorption, forming a covalent-like Au–S bond with the release of hydrogen, and the substantial bond strength (∼40–50 kcal/mol) far exceeds that of physisorption, enabling the formation of highly stable monolayers (Engel et al., 2017; Häkkinen, 2012; Pensa et al., 2012; Häkkinen, 2012). At the nanoscale, under-coordinated gold atoms on nanoparticle surfaces exhibit enhanced reactivity compared to bulk gold, increasing the density of accessible adsorption sites and promoting strong binding of thiol-containing biomolecules. The formation of self-assembled monolayers (SAMs) on gold electrodes has been foundational in engineering electrochemical interfaces (Tverdokhlebova et al., 2024; Vericat et al., 2010; Love et al., 2005). In biological systems, cysteine residues serve as the primary source of thiol groups; although many are involved in disulfide bonds, exposed or labile thiols can participate in Au–S binding under controlled incubation conditions. In electrochemical sensing, Au–thiol interactions are widely exploited for surface functionalization, biomolecule immobilization, receptor-free adsorption-based sensing, and interfacial passivation studies (Momeni et al., 2022). Surface passivation in electrochemical systems occurs when adsorbed species block active sites at the electrode–electrolyte interface, thereby reducing electron transfer and limiting electrochemical activity. Protein adsorption onto metallic electrodes results in steric hindrance, alteration of the electrical double-layer structure, increased charge-transfer resistance, and a consequent decrease in faradaic current. When a biomolecule adsorbs onto a nanostructured gold surface, it can form a partially insulating layer that reduces the effective electroactive surface area and slows interfacial electron-transfer processes.

In this study, we present a receptor-free electrochemical sensing strategy for CA 15-3 detection that is based on chemically mediated surface interactions rather than antigen–antibody recognition. The approach exploits the intrinsic affinity between gold and sulfur-containing functional groups, where accessible thiol groups in the biomarker undergo Au–S chemisorption at the nanostructured gold interface. This thiol-mediated adsorption leads to modulation of electron transfer at the electrode surface and progressive suppression of the intrinsic gold oxidation peak as analyte concentration increases, enabling a concentration-dependent electrochemical response under controlled conditions. While many plasma proteins contain cysteine residues, only surface-accessible thiols or labile disulfide bonds can effectively participate in interfacial interactions with gold, which underpins the sensing mechanism. Building on this principle, we developed a cost-effective and disposable sensing platform by modifying pencil graphite electrodes (PGEs) with gold nanoparticles (AuNPs). Compared to conventional gold electrodes, the AuNP-modified PGEs exhibit enhanced analytical performance, attributed to increased electroactive surface area and improved electron transfer kinetics provided by the nanostructured gold layer, resulting in improved sensitivity and lower detection limits. Additionally, the disposable nature of the platform eliminates the need for extensive electrode cleaning and allows the use of a freshly prepared AuNP–PGE electrode for each measurement, which minimizes cumulative surface fouling effects and ensures reproducible baseline responses. A previously reported multi-coating technique was employed to achieve rapid and uniform AuNP deposition, further improving reproducibility and fabrication efficiency (Ghaffari et al., 2023; Ghaffari et al., 2024). Overall, this work establishes a chemically driven sensing framework based on interfacial thiol–gold interactions and demonstrates the feasibility of label-free detection of thiol-containing biomarkers such as CA 15-3, providing a foundation for the development of simplified and cost-effective electrochemical sensing platforms.

Materials and apparatus

CA 15-3 was procured from Biocom, Africa. Chloroauric acid (HAuCl 4) was obtained from Sigma-Aldrich, United States (www.sigmaaldrich.com). Phosphate-buffered saline (PBS) was purchased from Sigma-Aldrich, United States (www.sigmaaldrich.com). Human serum plasma (sterile-filtered) was obtained from Sigma-Aldrich (St, Louis, MO, United States) and used as the biological matrix for real sample experiments. Pencil graphite electrodes (Pentel®, HB, 0.5 mm in diameter and 6 cm in length) were sourced from a local bookstore. Stock solutions of CA 15-3 at varying concentrations were prepared in PBS buffer. For electrochemical studies, PBS containing 5 mM [Fe(CN) 6 ] 3- / 4- was used as the supporting electrolyte. All reagents employed were of analytical grade, and PBS buffer solutions were used consistently throughout the experiments. Morphological and elemental characterization of the electrodes was performed using a scanning electron microscopy (SEM) equipped with energy-dispersive X-ray spectroscopy (EDS) (Phenom XL, Thermo Scientific, Waltham, MA, United States). Additionally, atomic force microscopy (AFM) was conducted using a Nanosurf Easyscan system to analyze the surface topography of the electrodes (Nanosurf AG, Liestal, Switzerland). Electrochemical measurements were carried out using a Biologic potentiostat (Clamart, France). The system was configured with a three-electrode setup: the working electrode consisted of either a gold electrode or pencil graphite electrodes modified with gold nanoparticles, an Ag/AgCl reference electrode (saturated KCl), and a platinum counter electrode.

Assembly strategy and evaluation of multi-electrode AuNPs-modified PGE

A 5 mM solution of Au 3+ was prepared by dissolving chloroauric acid (HAuCl 4) in 1 M H 2 SO 4. Polyethylene terephthalate (PET) containers with a capacity of 20 mL were used to fabricate custom multi-compartment electrochemical coating cell for the simultaneous modification of eight pencil graphite electrodes (PGEs). Each PET container lid was drilled with eight evenly spaced holes (0.7 mm diameter) around the circumference, which were fitted with metallic brackets serving as holders and electrical contacts for the PGEs. These eight individual PGE contacts were interconnected using 0.5 mm copper wire, forming a single unified working electrode system. Additionally, two central holes (5 mm and 3 mm in diameter) were drilled into the lids to accommodate Ag/AgCl reference and platinum wire counter electrodes, respectively, as illustrated in Figure 1. The entire multielectrode setup was used in combination with an external magnetic stirrer to facilitate uniform deposition. Prior to modification, the PGEs were cleaned by immersion in 3 M nitric acid, followed by thorough rinsing with deionized water. The cleaned electrodes were then electrochemically conditioned in 10 mL of 0.1 M acetate buffer by scanning from 0.0 V to 1.0 V using differential pulse voltammetry for three replicate cycles. Subsequently, the pencil-rod tips were immersed in 10 mL of the prepared Au 3+ solution for electrodeposition. Gold nanoparticles (AuNPs) were deposited simultaneously onto the eight PGEs by electrochemical reduction of Au 3+ under constant magnetic stirring. The deposition was carried out by first applying a fixed potential of −0.6 V for 3 min, followed by cyclic voltammetry from −1.0 V to +1.0 V for 20 successive cycles to promote uniform nanoparticle formation. A magnetic stir bar was placed in the solution to ensure effective hydrodynamic mixing during deposition. Instrumental parameters for the electrodeposition process were as follows: deposition time of 3 min, deposition potential of −0.6 V, equilibration time of 10 s, sweep rate of 0.05 V·s −1, and voltage step of 0.005 V. Following deposition, the AuNPs-modified PGEs were air-dried for 1 hour prior to electrochemical measurements.

Figure 2 depicts the modification process of the bare PGEs via electrodeposition of gold nanoparticles (AuNPs) onto their surfaces. After deposition, the AuNPs are visibly distributed on the electrode surface. To ensure reproducibility across measurements, a consistent 1.2 cm length at the tip of each modified PGE is exposed to the electrolyte by insulating the remaining portion with a layer of nail polish. This selective exposure guarantees that only the defined geometric electrode area (0.19 cm 2) participates in electrochemical measurements, while the rest of the immersed electrode is effectively insulated. Such standardization minimizes variability and ensures consistent and reproducible electrochemical responses across all modified electrodes used in the experiments.

Detection of CA 15-3 at AuNPs-PGE

To prepare the modified PGEs for use as working electrodes in CA 15-3 detection, a copper wire was affixed to one end of each pencil graphite rod to establish electrical contact with the potentiostat. The assembly was housed within a plastic syringe, where the PGE and copper wire were inserted such that a portion of the electrode tip protruded from one end of the syringe, while the copper wire exited through the opposite end (Figure 3). The electrochemical performance of the gold nanoparticle-modified pencil graphite electrode (AuNPs–PGE) for CA 15-3 detection was evaluated under optimized conditions. The AuNPs–PGE was incubated with CA 15-3 solutions for 25 min to enable effective interaction between the target protein and the electrode surface. After incubation, a potential of −0.5 V was applied for 20 s using chronoamperometry. This was followed by linear sweep voltammetry (LSV) to record the current responses. The resulting data were used to construct a calibration curve and determine the detection limit of the sensor for CA 15-3. All experiments were performed in triplicate (n = 3). Data are expressed as mean ± standard deviation. The limit of detection (LOD) was calculated using the 3σ/slope method.

Characterization

The surface characterization of the pencil graphite electrode (PGE) modified with gold nanoparticles (AuNPs) was performed using scanning electron microscopy (SEM) and atomic force microscopy (AFM). SEM analysis, equipped with energy-dispersive X-ray spectroscopy (EDS), was employed to investigate the surface morphology and confirm the presence of gold nanoparticles on the electrode surface. AFM was further used to examine the surface topography and assess the distribution of AuNPs on the PGE. For electrochemical characterization, the AuNPs-modified PGE (AuNPs-PGE), prepared as described in the experimental section, was employed as the working electrode for the electrochemical detection of CA 15-3 in this study. SEM and AFM analyses were interpreted in relation to the increase in electroactive surface area resulting from AuNP modification and the corresponding enhancement of the baseline gold oxidation current. Additionally, ferri/ferrocyanide measurements were employed solely to verify the successful deposition of AuNPs on the electrode surface and to confirm the associated improvement in electron-transfer kinetics.

Surface characterization of AuNPs-PGE

The surface morphology of the PGE modified with gold nanoparticles was characterized using scanning electron microscopy (SEM), as shown in Figure 4. The SEM images (Figures 4a1, b1) display the bare PGE and the gold nanoparticle-modified PGE at a magnification of ×550. The bare PGE surface exhibits surface roughness with machining-induced grooves along the polishing direction. After modification with gold nanoparticles, the surface appears uniformly coated with electrodeposited gold; however, higher magnification is required to observe the nanoparticles more distinctly. At 110,00× magnification (Figures 4a2, b2), the SEM images clearly reveal that the electrodeposited gold nanoparticles, formed from a 5 mM HAuCl 4 solution, are uniformly distributed as round-shaped particles across the electrode surface. Furthermore, the energy-dispersive X-ray spectroscopy (EDS) spectra (Figures 4a3, b3) confirm the successful deposition of gold nanoparticles, as indicated by the prominent Au peak observed in the modified PGE spectrum (b 3).

Figure 5 presents the AFM surface characterization of the PGE before and after modification with gold nanoparticles. At a scan area of 110 µm (Figures 5a1, a2), the bare PGE displays noticeable surface roughness with distinct groove patterns. After modification, the AFM image (Figure 5a2) shows that gold nanoparticles are distributed across the surface, covering both the grooves and the flatter regions. At a higher resolution (15 μm; Figures 5b1, b2), the electrodeposited gold nanoparticles, formed from a 5 mM HAuClO 4 solution, exhibit a predominantly spherical morphology with sizes below 100 nm. These AFM observations confirm the successful deposition and uniform distribution of gold nanoparticles on the PGE surface.

Electrochemical characterization of modified and unmodified PGE electrodes

Figures 6a,b illustrate the comparative electrochemical behavior of bare pencil graphite electrodes (PGEs) and those modified with gold nanoparticles (AuNPs), assessed using Nyquist impedance plots and cyclic voltammetry (CV), respectively. These measurements were carried out using a 5 mM solution of the [Fe(CN) 6 ] 3- / 4- redox couple in 0.1 M KCl, with frequency ranging from 0.01 Hz to 1300 Hz. The Nyquist plots showed that the charge transfer resistance (R ct) was effectively eliminated following modification of the PGE surface with gold nanoparticles. While the bare PGE showed a poorly defined semicircle with an estimated R ct of 508.3 Ω, the modified electrode exhibited no semicircle, indicating a near-complete elimination of charge transfer resistance. This behavior is attributed to the enhanced conductivity and increased electroactive surface area provided by the gold nanoparticles, which facilitate efficient electron transfer at the electrode–electrolyte interface.

Further quantitative analysis of the CV results using the Randles–Sevcik equation confirmed the enhancement in electrochemical performance. The bare PGE exhibited an electroactive surface area of 1.10 cm 2 with a roughness factor of 5.8, whereas the AuNP-modified PGE showed a substantially larger electroactive surface area of 4.21 cm 2 and a roughness factor of 22.3. This nearly fourfold increase demonstrates that AuNP modification significantly improves redox activity. As shown in Figure 5b, the CV profiles revealed that the bare PGE produced sluggish electron transfer with a large peak-to-peak separation (ΔE p) of 0.85 V, while the AuNP-modified electrode displayed sharper peaks with a smaller ΔE p of 0.116 V, indicating faster and more reversible electron transfer kinetics. Together, the EIS and CV analyses confirm that surface modification with AuNPs greatly enhances the electrochemical responsiveness and catalytic efficiency of the PGE. The key electrochemical parameters extracted from these measurements are summarized in Table 1, highlighting the substantial improvement in electron‐transfer kinetics and surface properties after gold nanoparticle modification.

Electrochemical behavior of gold electrode, bare PGE, and AuNPs-modified PGE in PBS

A comparison of the electrochemical responses of a conventional gold electrode, an unmodified pencil graphite electrode (PGE), and a PGE modified with gold nanoparticles (AuNPs) in phosphate-buffered saline (PBS) is shown in Figure 7. The gold electrode displays a well-defined oxidation peak at approximately 1.2 V, consistent with the characteristic oxidation of gold in neutral media. In contrast, the bare PGE exhibits negligible electrochemical activity under the same conditions, indicating limited surface reactivity in PBS. However, upon modification with gold nanoparticles, the PGE shows a pronounced anodic peak at around 1.2 V, mirroring the oxidation behavior observed with the gold electrode. This suggests successful deposition of AuNPs and the formation of a gold-active surface on the PGE. The enhanced peak current on the AuNPs-PGE reflects a significantly increased electrochemically active surface area, which is critical for applications relying on the interaction between gold and thiol groups, such as the detection of CA 15-3 biomarkers. Gold has a natural affinity for thiol groups, and the larger effective surface area of the AuNPs-PGE provides more binding sites for thiolated molecules compared to the flat gold electrode. Notably, the geometric surface area of the PGE is approximately 0.19 cm 2, compared to just 0.02 cm 2 for the gold electrode. When this larger area is functionalized with AuNPs, not only does the available gold surface increase substantially, but the high surface-to-volume ratio of the nanoparticles further enhances the electrode’s sensitivity. This modification approach offers several practical advantages: increased sensitivity, lower cost, and the potential for disposable, pre-functionalized electrodes without the need for extensive cleaning or maintenance.

Reproducibility of AuNPs-modified PGE in PBS

Figure 8 illustrates the reproducibility of the AuNPs-modified PGE in phosphate-buffered saline (PBS) in the absence of the target biomarker, CA 15-3. As shown in Figure 8a, the electrode demonstrates highly stable electrochemical behavior over five consecutive measurements using the same modified electrode, with negligible variation in peak current intensity. The relative standard deviation (RSD) for the peak current was calculated to be 0.4% (n = 5), indicating excellent reproducibility and consistency of the modified electrode under these conditions. This stability confirms that any subsequent changes observed in the peak current upon exposure to CA15-3 can be confidently attributed to the presence and concentration of the biomarker in solution. Therefore, the AuNPs–PGE provides a reliable baseline signal in PBS, reinforcing its suitability for sensitive detection of CA 15-3. Figure 8b shows the results obtained using a different, newly prepared modified electrode for each measurement. In this case, all electrodes were coated simultaneously using the multiple-coating technique. The results exhibit excellent reproducibility, with an RSD of 0.5% (n = 8), demonstrating the effectiveness and consistency of the simultaneous multiple-electrode modification approach for producing uniform and reliable sensor performance. In contrast, Figure 8c presents the results when each electrode was modified individually, rather than simultaneously. Although the same coating procedure was followed in principle, the electrodes were prepared one at a time, which introduced greater variability, likely due to small, uncontrolled differences in conditions or human handling during the electrodeposition process. As a result, the reproducibility was considerably lower, with an RSD of 2.7% (n = 6). These results confirm that the AuNPs–PGE can be used either as a single-use disposable sensor or reused for at least five consecutive measurements without compromising performance. Furthermore, when the multiple-coating technique is applied simultaneously to modify several electrodes, it yields consistent and reproducible results, greatly simplifying the fabrication process and enhancing the reliability of the sensor.

Analytical response of CA 15-3 biomarker towards the AuNPs–PGE

Detection of CA 15-3 was carried out in PBS by monitoring the intrinsic oxidation behavior of the gold-modified electrode. The detection strategy is based on the interaction between CA 15-3 and the AuNPs–PGE surface, where accessible thiol groups in the biomolecule exhibit strong affinity toward gold and promote adsorption at the electrode interface. Since the analytical response arises from suppression of the gold oxidation peak, adsorption of CA 15-3 leads to passivation of electroactive gold sites and hinders electron transfer at the electrode surface. As surface coverage increases with increasing CA 15-3 concentration, progressive attenuation of the gold oxidation peak is observed, reflecting the relationship between biomolecule adsorption and reduced electrochemical activity of the gold interface. In this context, the nanostructured gold layer provides a high-density, surface-sensitive platform that amplifies interfacial electrochemical responses, thereby enhancing the magnitude of the measurable signal and improving the dynamic range and overall sensitivity of the system, enabling quantitative detection of CA 15-3. This behavior was confirmed during the electrochemical study of CA 15-3 detection. The performance of the AuNPs–modified pencil graphite electrode (AuNPs–PGE) was evaluated using a combination of chronoamperometry (CA) and linear sweep voltammetry (LSV) under optimized conditions. Calibration studies were performed using CA 15-3 concentrations ranging from 1 to 6 ppb, with the corresponding voltammograms and calibration curve shown in Figure 9. This concentration range was selected to evaluate the sensor response within a low-concentration regime, where surface adsorption effects and changes in interfacial electron transfer are most sensitive and can be accurately resolved. Operating in this range also minimizes potential saturation of the gold surface and ensures a well-defined, concentration-dependent attenuation of the oxidation signal. These results represent the average of five independent experiments (n = 5), with a relative standard deviation (RSD) of less than 5%. A prominent oxidation peak at 1.2 V, attributed to gold oxidation, was observed to decrease consistently with increasing CA 15-3 concentration. A strong inverse linear relationship was obtained between peak current and CA 15-3 concentration, with a correlation coefficient of R 2 = 0.98 (n = 7). The calculated detection limit of the sensor was 0.12 ppb (0.00012 U/mL) using the 3σ/slope method, which is orders of magnitude lower than normal serum levels (<30 U/mL). Given that 30%–50% of breast cancer patients exhibit CA 15-3 concentrations exceeding 100 U/mL, this exceptionally low detection limit demonstrates the high sensitivity of the platform and supports its potential for detecting the CA 15-3 biomarker well below clinically relevant concentrations.

The analytical performance of the AuNPs–PGE sensor was compared with previously reported methods, as summarized in Table 2. The platform demonstrated comparable or superior sensitivity, while offering the advantages of shorter analysis time, lower cost, and the elimination of biological recognition elements. Notably, this approach allows for CA 15-3 detection using disposable, low-cost electrodes, making it a promising alternative for rapid and label-free biomarker sensing.

Interference study

The selectivity of the AuNP-modified pencil graphite electrode toward CA 15-3 detection was evaluated by examining the effect of several potential interfering species commonly present in human plasma. Linear sweep voltammetry (LSV) measurements were performed for the determination of 5 ppb CA 15-3 both in the absence and in the presence of possible interferents (Figure 10). The response obtained for CA 15-3 alone was taken as 100%, and the responses recorded in the presence of interferents were expressed as relative sensor responses (%), representing the magnitude of signal suppression compared to CA 15-3 alone. The selected interfering species were chosen based on their physiological presence in human serum and their chemical functionality, particularly the presence or absence of accessible thiol groups capable of interacting with gold surfaces. Two categories of interferents were investigated. The first group consisted of non-thiol-containing small molecules frequently found in plasma, including urea, glucose, and lactate. These compounds do not contain accessible thiol groups capable of forming strong chemisorption interactions with gold surfaces. The second group included species containing accessible thiol functionalities, specifically cysteine and human serum albumin. Albumin contains a well-known free cysteine residue (Cys34) that can interact strongly with metallic surfaces, while cysteine represents a small-molecule thiol that readily forms Au–S bonds. As shown in Figure 10, the presence of a two-fold excess concentration (10 ppb) of non-thiol-containing interferents (urea, glucose, and lactate) produced negligible changes in the voltammetric response of the sensor toward the detection of 5 ppb CA 15-3. The gold oxidation peak current remained essentially unchanged, indicating that these species do not significantly interact with the gold nanoparticle surface under the experimental conditions used. This observation suggests that non-thiol-containing analytes do not contribute appreciably to surface passivation of the electrode. In contrast, the presence of thiol-containing interferents, namely albumin and cysteine, resulted in a pronounced change in the electrochemical response of the sensor. A noticeable increase in signal suppression was observed in the presence of these species, indicating their ability to interact strongly with the gold nanoparticle-modified surface. The observed effect is attributed to the formation of stable Au–S bonds between accessible thiol groups and the gold nanoparticle surface. These results support the proposed thiol chemisorption-driven detection mechanism. Thiol-containing analytes exhibit stronger and more persistent surface adsorption due to the well-established thermodynamic stability of Au–S bonds. In the case of CA 15-3, accessible cysteine residues within the MUC1-derived glycoprotein fragment can interact with the gold nanoparticle surface, resulting in progressive surface passivation and attenuation of the gold oxidation peak as the analyte concentration increases. Similar behavior observed with albumin and cysteine further confirms that the sensing response is governed primarily by thiol-mediated adsorption processes.

Overall, the interference study demonstrates that the developed sensing platform exhibits selective responsiveness toward thiol-containing biomolecules. While non-thiol plasma components do not significantly influence the electrochemical signal, thiol-bearing species can interact with the gold surface and contribute to surface passivation. These findings provide mechanistic support for the Au–thiol interaction-based sensing strategy employed for the electrochemical detection of CA 15-3.

Analysis of human serum plasma by the AuNPs–PGE

The applicability of the AuNPs–PGE sensor for the determination of CA 15-3 in human serum plasma was evaluated using the standard addition method. Measurements were carried out in triplicate on samples spiked with 2 ppb CA 15-3. A strong inverse linear relationship between peak current and CA 15-3 concentration was observed, with a correlation coefficient of 0.999 (n = 3). Recovery values ranged from 97.5% to 104%, demonstrating satisfactory accuracy (Table 3). The relative standard deviation (RSD) for the measurements ranged from 1.74% to 2.77%, indicating high precision with less than 3% variation across replicate determinations.

The applicability of the AuNPs–PGE sensor for the determination of CA 15-3 in human serum plasma was evaluated using the standard addition method. Plasma samples were diluted with phosphate-buffered saline (PBS) prior to analysis to reduce matrix complexity and minimize nonspecific adsorption of plasma proteins on the electrode surface. Measurements were performed in triplicate using plasma samples spiked with 2 ppb CA 15-3. As demonstrated in the interference study, thiol-containing plasma species can contribute to suppression of the gold oxidation peak through Au–S chemisorption. In the diluted plasma matrix, these components generate a stable background level of surface passivation that serves as the baseline electrochemical response. Upon spiking the samples with increasing concentrations of CA 15-3, additional attenuation of the gold oxidation peak was observed, indicating further surface passivation caused by increased thiol-mediated adsorption on the AuNP-modified electrode. This incremental suppression relative to the matrix baseline enabled quantitative determination of CA 15-3 using the standard addition approach. A strong inverse linear relationship between the gold oxidation peak current and CA 15-3 concentration was obtained, with a correlation coefficient of 0.999 (n = 3). Recovery values ranged from 97.5% to 104%, demonstrating satisfactory analytical accuracy (Table 3). The relative standard deviation (RSD) ranged from 1.74% to 2.77%, indicating good reproducibility across replicate measurements. These results confirm that although plasma proteins introduce a background level of surface passivation, increasing concentrations of CA 15-3 produce measurable additional suppression of the gold oxidation peak, enabling reliable quantification of the analyte in diluted human plasma.

Summary

Pencil graphite electrodes (PGEs) modified with gold nanoparticles (AuNPs) have demonstrated successful label-free electrochemical detection of the breast cancer biomarker CA 15-3 in both phosphate-buffered saline (PBS) and human plasma samples. This strategy eliminates the need for biological recognition elements, such as antibodies or aptamers, simplifying the sensing platform and enabling cost-effective, single-use disposable electrodes. The disposable nature of PGEs also removes the necessity for cleaning and maintenance associated with reusable systems. Recent studies further confirm that PGEs remain highly topical, with applications ranging from sensitive detection of epigenetic biomarkers in urine (Bilge et al., 2025), improved paracetamol sensing via surface modification (Korkmaz et al., 2025), drug–DNA interaction studies with daptomycin (Aslan and Levent, 2024), to nepafenac detection in pharmaceuticals and biological samples (Ersali and Levent, 2024). These works highlight PGEs as low-cost, eco-friendly, and versatile tools for biomedical and pharmaceutical applications. A notable limitation of PGEs is their mechanical fragility, which can make handling difficult. To address this, one approach involves crushing the graphite and reconstituting it into pellet or tablet-like electrodes using a binding agent such as potassium bromide (KBr). Another promising strategy is blending pencil graphite with gold nanoparticles in a carbon paste formulation suitable for screen-printed electrodes, enhancing both electrochemical performance and mechanical robustness. Future work could explore these fabrication routes to enhance the mechanical stability of the sensor platform. Nonetheless, AuNP-modified PGEs have proven to be an effective and reliable platform for sensitive, label-free detection of CA 15-3 without requiring any biological recognition elements.

Study limitations

The sensing strategy presented here relies on adsorption-mediated modulation of gold surface oxidation behavior rather than molecular recognition elements. While this receptor-free approach enables label-free detection and simplified electrode preparation, it does not provide antigen–antibody specificity. The sensing response is governed primarily by thiol-mediated adsorption at the gold nanoparticle surface; therefore, the platform should be regarded as chemically selective toward thiol-containing species rather than immunochemically specific for CA 15-3. Although the analytical performance demonstrates proof-of-concept feasibility under the conditions investigated, further studies in more complex biological environments would be required to fully assess its potential for clinical applications.

In this study, a simple and cost-effective electrochemical approach was developed for the detection of CA 15-3 without the use of biological recognition elements, relying instead on the natural affinity between gold and the thiol groups in CA 15-3. Pencil graphite electrodes (PGEs) were modified with gold nanoparticles (AuNPs) using a rapid multi-electrode coating technique, enabling uniform deposition and reproducible performance. The resulting AuNPs–PGEs showed enhanced sensitivity compared to conventional gold electrodes, achieving a detection limit of 0.12 ppb for CA 15-3. This platform demonstrated excellent reproducibility, low cost, and disposability, offering a promising tool for label-free detection of thiol-containing biomarkers and potential early-stage cancer diagnostics.