Electrochemical biosensing of sperm protein 17 in clinical samples using a hydrothermally synthesized MoS₂@rGO@MWCNTs ternary system without prior pre-treatment

Abstract

Sperm protein-17, also known as Sp17, is a testis-linked cancerous antigen found in various cancers, making it a potential biomarker for cancer detection. However, detecting Sp17 in human serum is challenging due to its low concentration and the absence of sensitive, specific detection methods. This study presents a fast, accurate, reliable, and cost-effective biosensor for Sp17 detection, utilizing a disposable carbon cloth (CC)-based electrode modified with a ternary nanosystem of molybdenum disulfide (MoS₂), reduced graphene oxide (rGO), and multi-walled carbon nanotubes (MWCNTs). The ternary nanosystem was prepared through a simple hydrothermal approach and characterized using various techniques, including X-ray diffraction, transmission electron microscopy, scanning electron microscopy, Raman spectroscopy, and electrochemical studies, which confirmed its robust formation and suitability for Sp17 sensing. The nanosystem exhibited electrocatalytic activity, biocompatibility, and stability. A Sp17-specific antibody (anti-Sp17) was attached to the surface of the electrode, and the immunosensor showed a linearity range from 0.01 to 11 ng mL⁻¹ with a detection limit (LOD) of 0.23 ng mL⁻¹ as well as a limit of quantification (LOQ) of 0.79 ng mL⁻¹, achieving a sensitivity of 9.97 μA ng mL⁻¹ cm². The mechanism behind the sensing is attributed to charge transfer-induced shifts in the Fermi level, causing band bending at the interface between the nanosystem and the target molecule, leading to enhanced sensitivity. The theoretical analysis correlates these Fermi energy shifts with device sensitivity, particularly with antibody immobilization. The immunosensor was successfully used to detect Sp17 in human serum samples, showing good accuracy, reproducibility, and selectivity. It also correlated with a commercial enzyme-linked immunosorbent assay (ELISA), suggesting its potential for clinical diagnostics, particularly for ovarian and other Sp17-related cancers.

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1. Introduction

Nanotechnology development has shifted the focus of the scientific community away from traditional sensing methods and led to the development of highly specific biosensors capable of detecting bioanalytes at nanomolar levels.¹ However, electrochemical determination of cancer biomarkers in bare electrodes, such as glassy carbon electrodes (GCEs), indium tin oxides (ITO), or screen-printed electrodes (SPEs), often results in poor sensitivity levels due to the potential fouling of the electrode surface caused by absorbed oxidized residues. Thus, an electrode surface modified with nanomaterials (NMs) facilitates selective and sensitive detection performance by providing a larger surface area and faster electron transfer rates.

In recent times, NMs have been used to develop biosensors to enhance the analytical characteristics of these devices for detecting chemical/biological substances. Over the past few years, there has been a growing interest in single-layer or few-layer two-dimensional transition metal sulfides, including titanium disulfide (TiS₂),² tungsten disulfide (WS₂),³ molybdenum diselenide (MoSe₂),⁴ molybdenum disulfide (MoS₂),⁵ and tungsten diselenide (WSe₂),⁶ which have garnered growing interest from the academic society. Among them, molybdenum 2D disulfide nanosheets (MoS₂ NSs) have gained interest in the sensing industry because of their distinct mechanical, biological, physicochemical, and electrical properties.⁷ MoS₂ NSs consist of triple layers of S–Mo–S with widely recognized semiconducting characteristics found in metal dichalcogenide materials.⁸ The exceptional electrochemical characteristics and luminescent capabilities of MoS₂-based NMs have established them as innovative biosensing probes for precisely detecting various analytes.⁹ Their multidimensional architectures are the main reason for their appeal due to their versatile potential for multiple applications. MoS₂ NSs are highly intriguing for the advancement of freshly developed biosensors, not only because their surface can be functionalized with thiolated molecules^10,11 but also because they can create new platforms with a larger comparative surface area, resulting in enhanced sensitivity. Several biosensors utilizing these NMs have been reported in the literature^12–17, but this field is still in its early stages and requires further exploration.

Nevertheless, pristine MoS₂ NSs suffers from a limited number of available active sites, low conductivity, and the tendency for aggregations to restack, all of which impede electron transport.¹⁸ Adding a second component for making binary hybrids is viewed as a highly effective approach to address these problems, as outlined in our previous work.¹⁵ Several low-dimensional NMs, such as graphene,¹⁹ carbon nanotubes (CNTs),²⁰ carbon dots (CDs),²¹ graphene quantum dots,²²etc., have been employed to enhance the biosensing capabilities of MoS₂ NSs by forming hybrid structures.^23,24 Therefore, in this work, we have chosen to take a further step by utilizing MoS₂ NSs in conjunction with reduced graphene oxide (rGO) and multiwalled carbon nanotubes (MWCNTs) for ternary nanosystem-based electrochemical immunosensor development to identify a cancer biomarker called sperm protein 17 (Sp17). MoS₂ NSs's exceptional electronic properties, when combined with the high conductivity of rGO and MWCNTs, significantly enhance the rapid electron transfer and, thus, the sensitivity of the detection platform. This heightened sensitivity is paramount for detecting minute concentrations of biomarkers indicative of cancer presence. Moreover, the large surface area afforded by MoS₂ NSs, rGO, and MWCNTs facilitates efficient biomolecule immobilization, enabling precise capture and selective detection of cancer biomarkers, which is crucial for distinguishing specific biomarkers amidst complex biological matrices.

Classical disk-shaped GCE, SPE, and ITOs are commonly employed as substrates for fabricating electrochemical biosensors. Nevertheless, surface modification can sometimes lead to additional disadvantages, such as reduced biocompatibility and conductivity of the coating materials, or necessitate intricate methods to include appropriate functional groups onto their surfaces.²⁵ Hence, developing electrode platforms using easy electrode alteration techniques and employing a solitary biocompatible substance is essential. These factors are important in the battle against cancer, making it highly advantageous to develop innovative methods to develop inexpensive and extremely sensitive devices for detecting cancer biomarkers. Even though the determination of cancer biomarkers has been reported using ITO, GCE, or SPEs, a sensing platform that uses flexible carbon cloth (CC) has not been reported to the best of our knowledge. CC is an alternative to the conventional carbon paste SPE and GCE that utilize electrochemical-based methodologies.²⁶ CC typically consists of polyacrylonitrile-based carbon fiber, which possesses exceptional flexibility, adjustable size properties, and high conductivity with high porosity.^26,27 CC is utilized in various electrochemical-based methods, including supercapacitors, fuel cells, and applications in sensors and biosensors.^28,29

Cancer, being a very lethal illness, poses a significant threat to human life and imposes a substantial burden on society.^30,31 The outcome of cancer treatment is intricately linked to the stage of cancer, with a higher likelihood of being cured observed in patients with early-stage disease.³² Therefore, early detection of cancer is of utmost importance. Different circulating biomarkers in human bodily fluids can offer valuable insights for initial cancer diagnosis, and their aberrant levels can be used to evaluate the advancement of the disease.^33,34 Sp17, a cancer-testis antigen (CTA) family member, is a conserved mammalian protein of human and animal testis, and spermatozoa. While primarily expressed in healthy male spermatozoa and embryonic germinal cells, its expression extends to cancerous tissues, as extensively documented in various studies across multiple cancer types, including ovarian, nervous system, and esophageal squamous cell cancers.³⁵ Several highly valid techniques have been developed to detect cancer biomarkers, such as enzyme-linked immunosorbent assay (ELISA) to detect protein biomarkers and polymerase chain reaction (PCR) to identify nucleic acid biomarkers.³⁶ However, the inherent limitations of ELISA and PCR, such as the need for large equipment, skilled operators, and a laboratory environment, hinder their use in point-of-care (POC) diagnostics.³⁷ Thus, there is a strong need to develop innovative diagnostic technologies that can tackle these barriers.

This article presents a biosensor, i.e., an antibody-based electrochemical sensor that utilizes flexible CC as the primary substrate for Sp17 cancer biomarker determination. The CC is modified with 3D structures of a unique ternary nanosystem consisting of MoS₂@rGO@MWCNTs. The incorporation of MWCNTs can inhibit the formation of clusters in rGO NSs and MoS₂ NSs. The rGO NSs function as a path for the transmission of carriers and as an interface for the development of MoS₂ NSs. In addition, the composite material's 3D nanostructure offers many effective sites for immobilizing biorecognition molecules on the substrate surface through covalent bonds. This creates a stable platform that allows the binding of Sp17 to anti-Sp17 antibodies and its recognition. This detection is achieved by utilizing the changes in electrochemical responses caused by the [Fe(CN)₆]^3−/4− redox species. These sites contribute to the electron transmission and improve the MoS₂@rGO@MWCNT nanohybrid's electrochemical sensing properties. The biosensor described in this work measures the Sp17 protein concentration in phosphate buffer saline (PBS) and serum samples from patients. Moreover, a detailed analysis, supported by a schematic illustration, is provided for explaining the sensing mechanism for charge transfer-induced shifts in the Fermi energy, on the order of sub-μeV, and the resulting band at the electrode interface of the semiconductor. A theoretical study was conducted to show the relations of these Fermi energy shifts with the device's sensitivity, particularly in response to cancer cell immobilization at the molecular level. The findings demonstrate that the biosensor, based on the MoS₂@rGO@MWCNT nanosystem, exhibits a strong response, excellent selectivity, remarkable stability, and rapid reaction to Sp17 at room temperature (RT).

2. Experimental section

2.1 Chemicals and reagents

This section, with a detailed list of the materials utilized in this study, has been given in the ESI.† All reagents and chemicals employed were of analytical grade and utilized without additional treatments. Electrochemical analyses were conducted using Milli-Q water (resistivity: 18 MΩ cm⁻¹; Millipore, USA). Additionally, the Instrumentation section, which describes the advanced instruments employed to characterize the ligands, complexes, and ternary nanosystems synthesized in this work, has also been provided in the ESI.†

2.2 Synthesis of GO, MoS₂, and MoS₂@rGO

Graphene oxide (GO) was prepared using graphite powder along with concentrated HNO₃ and H₂SO₄via a modified Hummer's technique, with a minor modification. The synthesis methods for synthesizing MoS₂@rGO, MoS₂, and rGO from graphite have been described in our previously published work.¹⁵

2.3 Synthesis and functionalization of the MoS₂@rGO@MWCNT ternary nanosystem

The choice of carbon substrates is driven by their exceptional electrical conductivity and mechanical properties, which are advantageous for biosensor applications involving hybrid nanostructures. The synthesis parameters were adopted from a previously reported protocol to grow MoS₂ NSs on carbon substrates, specifically rGO and MWCNTs, with minor modifications to suit the current biosensor fabrication.³⁸ In summary, to synthesize the MoS₂@rGO@MWCNTs nanosystem, 100 mg of MWCNTs and 140 mg of GO powder were dispersed in 35 mL of deionized water (DI) and subjected to bath sonication for 3 h to exfoliate the multilayer GO stacked sheets. Separately, in another container, 2.28 g of NH₂CSNH₂ and 1.236 g of Na₂MoO₄·2H₂O were added in DI using a magnetic stirrer until a transparent solution formed. This solution was combined with the pre-dispersed GO and MWCNTs solution, which was continuously agitated for 1 h. The resultant reaction mixture was enclosed in an autoclave coated with Teflon and exposed to heat for the next 24 h in a furnace at a temperature of 220 °C. During hydrothermal treatment, the NH₂CSNH₂ aided in the reduction of GO, converting GO into rGO. Once the reaction was finished, the hydrothermal vessel was left to cool to 25 °C. After purification procedures, the final product was then collected by centrifugation at 10 000 rpm with deionized water and ethanol. Lastly, the MoS₂@rGO@MWCNT nanosystem was dried overnight in an oven at 80 °C to yield a black residue. Scheme 1(a) concisely illustrates the MoS₂@rGO@MWCNT nanohybrid synthesis process. During hydrothermal treatment, GO is reduced to rGO, MWCNTs remain intact, and MoS₂ is formed in situ from the reaction between sodium molybdate and thiourea. Thiourea is used in excess as both the sulfur source and a mild reducing agent. Assuming the complete conversion of molybdenum and sulfur precursors to MoS₂ and using the theoretical stoichiometry and molecular weights, the estimated weight ratio of the final composite components is approximately MoS₂ : rGO : MWCNTs = 1202 mg : 140 mg : 100 mg, which corresponds roughly to: ∼82% MoS₂: 9.5% rGO: 6.8% MWCNTs (by weight). In addition, the average yield of the nanomaterial is MoS₂: 78.5 ± 2.1%; MoS₂@rGO: 73.2 ± 1.8%; and MoS₂@rGO@MWCNTs: 69.4 ± 2.3%. The yields were determined by weighing the dried product after completion of the synthesis and purification steps, relative to the total mass of starting materials used.

Scheme 1 (a) Illustration depicting the synthesis and functionalization process of the MoS₂@rGO@MWCNT nanohybrid; (b) schematic of the working of the EPD process; and (c) schematic illustrating the procedures for fabricating the Sp17 immunosensor using an APTES/MoS₂@rGO@MWCNT modified CC electrode.

2.3.1 Functionalization of the MoS₂@rGO@MWCNT nanosystem. A linker was used to introduce functional groups on the nanohybrid surface for bioconjugating the bioreceptors. In this case, an amino-functionalized silane, i.e., 3-aminopropyltriethoxysilane (APTES), was used. The APTES, another end comprising the triethoxy group, undergoes hydrolysis, allowing the –NH₂ located on the other side of it to bind to the matrix surface, making APTES an effective and promising linking agent. 100 mg of the MoS₂@rGO@MWCNT nanosystem was introduced into isopropanol (20 mL) and treated using sonication to form a well-dispersed mixture. Afterward, 200 μL (98%) APTES was combined and agitated for 48 h at a speed of 300 rpm at 25 °C. Additionally, the resulting residue was washed with ethanol and Milli-Q water to eliminate unattached APTES molecules. This was achieved through the process of ultracentrifugation. The functionalized sample (APTES/MoS₂@rGO@MWCNTs) was dried at 60 °C. The washed material was stored at RT until it was needed for future usage.

2.3.2 Preparation of the APTES/MoS₂@rGO@MWCNT nanosystem dispersion. Aliquots of the APTES/MoS₂@rGO@MWCNT nanosystem were prepared in DI, followed by ultrasonication at a 2 mg mL⁻¹ concentration. These aliquots were subsequently used to modify the working electrode for further electrochemical investigations.

2.4. Uniform deposition of the functionalized ternary nanosystem on a CC electrode

CC was prepared as the base for nanoplatform fabrication, with its surface undergoing pre-treatment before MoS₂@rGO@MWCNT deposition. A large piece of CC was treated in a mixture of concentrated H₂SO₄ and HNO₃ (1 : 3) at 80 °C for 5 h to enhance surface reactivity, followed by thorough rinsing with DI and ethanol and drying at 80 °C for 6 h.^29,39 The acid treatment improves the reactive functional group's availability on the CC surface. After activating the surface, the CC was cut into the desired dimensions (0.5 × 1.5 cm) for MoS₂@rGO@MWCNT deposition using the electrophoretic deposition (EPD) technique. An APTES functionalized nanohybrid of MoS₂@rGO@MWCNTs was applied onto the pre-treated CC electrode utilizing an EPD process employing the GeNei equipment. EPD is a versatile method for producing thin coatings on electrically conductive substrates.⁴⁰ Nearly all nano-objects exhibit surface charges when dissolved, leading to a highly stable, soluble solution. When the solution is treated with an electric field, the charged units in both the aqueous/non-aqueous suspensions could be attracted to an electrode with an opposite charge [Scheme 1(b)]. This movement is caused by the applied electric field.⁴¹ This technique is rapid, uniform, well-suited for large-scale manufacturing, and permits versatility in the design of electrodes without the need for binders. Due to its numerous benefits, EPD has been extensively studied for its possible use in biosensors and fuel cells.

2.5 Fabrication procedure for the biosensing platform

The solution comprising anti-Sp17 antibodies (50 μg mL⁻¹) was made using PBS with a pH of 7.0. A total of 15 μL of anti-Sp17 was combined with 0.4 M EDC (activator, 7.5 μL) and 0.1 M NHS (coupling agent, 7.5 μL). Afterward, the pre-incubation of the solution (30 μL) was done for 40 min and then evenly applied on the APTES/MoS₂@rGO@MWCNTs/CC electrode using the drop-casting method. The antibody-immobilized electrode was stored in a humid compartment at 25 °C and then rinsed with PBS to eliminate unattached antibody molecules. The carboxyl group (–COOH) of the anti-Sp17 antibodies can form a strong amide bond (–OC–NH) by covalently binding with the amino terminus (–NH₂) of APTES. Finally, a 2% bovine serum albumin (BSA) solution was applied to the electrode to prevent non-specific binding to its active sites. The BSA/anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC immunoelectrode was kept at a temperature of 4 °C when not further used. Scheme 1(c) illustrates the sequential developmental process of the BSA/anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC immunosensor, including the biochemical reaction.

2.6 Collection and serum sample processing

Biological samples were collected following the institution's approved protocols to evaluate the developed immunosensor's effectiveness in detecting Sp17 protein in real samples. Ethical approval for patient sample collection was obtained from the Jawaharlal Nehru University (JNU) and the All India Institute of Medical Sciences (AIIMS), New Delhi. Patients provided written consent before blood collection. Serum samples were collected in pre-sterilized containers and refrigerated for 20 min to allow coagulation. The serum was subsequently put into 5 mL centrifuge tubes, washed for 15 min at 5000 rpm, and the supernatant obtained was further preserved at −20 °C. Finally, the serum aliquots were prepared and kept at −80 °C to avoid freeze–thaw cycles, ensuring sample integrity for future use.

3. Results and discussion

The ternary nanosystem-based biosensor follows a fundamental biosensing principle and involves several fabrication stages, as outlined in Scheme 1(a) and (b). Initially, MoS₂@rGO@MWCNT nanohybrids are synthesized using a single-step hydrothermal process and APTES functionalization. These APTES/MoS₂@rGO@MWCNT nanohybrids serve as immobilization matrices for anti-Sp17 antibodies. The antibodies are then attached to the APTES-coated surface via EDC/NHS chemistry (step 1). Subsequently, the APTES/MoS₂@rGO@MWCNT platform provides an appropriate modification surface for anti-Sp17 immobilization (step 2). Free carboxyl groups are blocked using BSA to minimize non-specific attachment, enhancing the interaction between antibodies and their Sp17 antigens (step 3). Finally, the prepared BSA/anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC immunoelectrode is employed for measuring Sp17 levels (step 4).

3.1 Protein–protein interaction and networking analysis with Sp17

Sp17, an immunogenic protein, belongs to the CTA family. It is a highly preserved mammalian protein in the spermatozoa and testis of both animals and humans,^42,43 comprising 151 amino acids [Fig. 1(b)] and possessing an apparent molecular mass of 24.5 kDa.⁴⁴ Sp17 helps control the process of acrosomal reaction, capacitation, and sperm maturation, as well as its relationship with the egg zona pellucida upon fertilization in normal tissues.⁴⁵ The biological process enrichment of Sp17 is shown in Fig. 1(a).

Fig. 1 (a) The STRING network showing the biological process (gene ontology) enrichment; (b) amino acid sequence of Sp17; (c) the STRING networking demonstrated by colored lines that occurred between the proteins, which indicate various interaction evidence types; (d) GO annotation showing the sub-cellular location of Sp17; and (e) 3D structure of Sp17 protein.

Furthermore, to identify potential interactions between Sp17 and serum proteins, we used the STRING database as a tool (https://string-db.org/, version 9.1) for retrieving interacting genes and proteins. Given the complex composition of serum proteins, analyzing Sp17's interactions with those implicated in cancer progression was prioritized. The STRING database integrates indirect (functional) and direct (physical) interactions between proteins, offering a comprehensive overview of these connections. As illustrated in Fig. 1(c), Sp17 is central in a densely connected network. From these findings, the selection of interfering proteins was made for additional testing to assess the proposed immunosensor selectivity. Fig. 1(d) shows the gene ontology (GO) annotation of Sp17 used in bioinformatics to assign standardized biological terms to genes and gene products (like proteins or RNA) to describe their functions, cellular locations, and roles in biological processes. The 3D structure of Sp17 has been shown in Fig. 1(e) (https://www.uniprot.org/uniprotkb/Q15506/entry#structure).

3.2 Structural and morphological characterization of nMoS₂, nMoS₂@rGO, and nMoS₂@rGO@MWCNTs nanohybrids

3.2.1 Raman analysis. The Raman tests gave additional information on the structural vibrations of the MoS₂, MoS₂@rGO, and MoS₂@rGO@MWCNTs. These findings are presented in Fig. 2. Fig. 2(a) and (d) display the MoS₂ NS Raman spectra, illustrating two phonon vibrations at 406 and 379 cm⁻¹ that correlate to the A¹_g MoS₂ out-of-plane as well as E¹_2g in-plane modes, respectively. The characteristic peaks observed in Fig. 2(b) and (c) correspond to MoS₂@rGO and MoS₂@rGO@MWCNT hybrid nanostructures, respectively. Monolayer MoS₂ typically exhibits distinct vibrational modes around 374 cm⁻¹ and 401 cm⁻¹. The frequency difference between these modes serves as a reliable parameter for estimating the number of MoS₂ layers within the material structure.⁴⁶Fig. 2(e) and (f) depict the as-synthesized nanohybrid Raman spectra covering a distinct range of wavenumbers. The peaks found at 1583 and 1347 cm⁻¹ in the MWCNTs and rGO are distinctive and represent the G and D bands of MWCNTs and graphene, respectively. Additionally, carbanion materials, like graphene, carbon fibers, or MWCNTs, exhibit distinct Raman peaks that are primarily associated with the G as well as D bands at around 1590 and 1360 cm⁻¹, respectively, and the degree of graphitization significantly affects these distinctive characteristics.⁴⁷ The ID/IG ratios for MoS₂@rGO@MWCNTs besides MoS₂@rGO have been determined as 1.03 and 1.01, respectively. The slight increase in the ratio upon MWCNTs incorporation indicates a marginal rise in structural defects, likely due to enhanced interfacial interactions and hybridization effects, which may contribute positively to the electrochemical performance of the composite.

Fig. 2 Raman plots of MoS₂ (a) and (d), MoS₂@rGO (b) and (e), and MoS₂@rGO@MWCNT nanohybrids (c) and (f) in various wavenumber ranges.

3.2.2 XRD analysis. X-ray diffraction (XRD) plots of all the materials were studied and presented in Fig. 3. Fig. 3(a) illustrates the XRD of MoS₂ NSs made using a simple hydrothermal process to examine the crystalline and structural characteristics. The diffraction peaks observed at 2θ, i.e., 13.2°, 32.8°, and 57.3°, correlate to the (002), (100), and (110) reflection planes, respectively. These peaks are distinctive to MoS₂ NSs and have been accurately indexed with the established MoS₂ structure. The reflection planes observed from the (002) plane correlate with the 0.33 nm d-spacing value, consistent with the MoS₂ 2H crystal structure. Additional evidence indicates the successful formation of hexagonal MoS₂ NSs with a significant distance between layers, as indicated by distinct peaks at 37.3, 48.4, and 69.5, which correspond to the (103), (105), and (201) planes, respectively (JCPDS No. 37-1492).^15,16

Fig. 3 XRD plots of (a) MoS₂ NS, (b) MoS₂@rGO, and (c) MoS₂@rGO@MWCNT nanohybrids.

Fig. 3(b) and (c) display the XRD spectra of the MoS₂@rGO and MoS₂@rGO@MWCNTs nanohybrid structures, respectively. The XRD plots exhibit similarities with the MoS₂ nanostructure, with a minor variation. The peaks at 13.5°, 32.9°, 39.1°, 48.7°, 58.42°, and 69.2°, as shown in Fig. 3(b) and (c), provide evidence for the existence of MoS₂ NSs in the hybrid framework. Both Fig. 3(b) and (c) exhibit a distinct peak with low intensity at a 27.8° angle corresponding to the (002) reflection plane of rGO. In contrast, this peak was slightly increased in intensity, as illustrated in Fig. 3(c), potentially due to the inclusion of MWCNTs in the hybrid. Furthermore, an additional distinct peak of MWCNTs originating from the (100) reflection planes can be observed at 42.5° in Fig. 3(c). The peak intensities resulting from MWCNTs and rGO are considerably lower than those of MoS₂ NSs due to the decreased crystallinity of both MWCNTs and rGO. Moreover, the reduced intensity indicates that rGO sheet restacking is constrained due to forming a hybrid structure.⁴⁸

3.2.3 XPS analysis. An X-ray photoelectron spectroscopy (XPS) examination has been conducted to determine the bonding modes and chemical composition of the synthesized materials. Fig. 4(a) depicts the comprehensive XPS scan of the MoS₂@rGO@MWCNTs nanohybrid. The peaks observed in Fig. 4(a) mainly indicate the presence of O, C, S, and Mo elements and some incidental contaminants in the sample. The C 1s curve results from graphite-like sp²-hybridized C, whereas the presence of oxygen-comprising functional groups causes the O 1s curve. Fig. 4(b) depicts the Mo XPS spectrum, demonstrating the existence of a doublet, i.e., 227.9 and 225.9 eV, owing to the 3d_3/2 and 3d_5/2 Mo⁴⁺ oxidation states, respectively.⁴⁹ Additional low-intensity peaks at 228.7 eV and 231.8 signify the presence of the Mo⁶⁺ oxidation state, specifically in the 3d_5/2 and 3d_3/2 orbitals, respectively. In addition, the C 1s deconvoluted peak consists of several energy peaks, as seen in Fig. 4(c). The prominent peak at 284.2 eV is attributed to graphitic carbon, whereas the peaks at 285.01 and 283.7 eV correspond to the C=O and C–OH configurations, respectively. The less intensified peak of oxidized C, i.e., (C=O and C–O), indicates that most oxygen-related groups were eliminated over the rigorous exfoliation process, resulting in GO reduction into rGO.⁵⁰ The occurrence of the sulfur 2p state is responsible for the photoelectron peak observed at 161.5 eV, as depicted in Fig. 4(d). In addition, the peaks at 161.5 and 161.9 eV are ascribed to the 2p_3/2 (S²⁻), and 2p_3/2 (S₂²⁻) atomic states of S, respectively. Furthermore, the most substantial peak at 162.7 eV, which has a significant magnitude, is associated to 2p_3/2 with the S⁶⁺ oxidation state.⁵¹

Fig. 4 XPS survey spectrum of the MoS₂@rGO@MWCNT nanohybrid (a); and deconvoluted plot of Mo(3d), C(1s), and S(2p) in the MoS₂@rGO@MWCNT nanohybrid (b)–(d).

3.2.4 FT-IR analysis. The Fourier transform-infrared spectroscopy (FT-IR) spectra of MoS₂, MoS₂@rGO, and MoS₂@rGO@MWCNTs were analyzed to confirm the successful synthesis and identify the functional groups present in each material, as shown in Fig. 5. In the FT-IR spectrum of pure MoS₂, a prominent peak appears around 470–500 cm⁻¹, attributed to the Mo–S stretching vibrations, confirming the characteristic structure of MoS₂. For the MoS₂@rGO composite, additional peaks corresponding to the functional groups of rGO are observed. A broad peak near 3400 cm⁻¹ is assigned to O–H stretching vibrations, indicating residual hydroxyl groups on the rGO surface. In contrast, the peak at approximately 1620 cm⁻¹ corresponds to C=C stretching, characteristic of the rGO structure. Another peak around 1050 cm⁻¹, attributed to C–O stretching, further confirms the presence of rGO in the composite. The characteristic Mo–S peak (470–500 cm⁻¹) of MoS₂ is retained, indicating successful integration of MoS₂ with rGO.

Fig. 5 FT-IR spectra of MoS₂, MoS₂@rGO, and MoS₂@rGO@MWCNTs.

In the MoS₂@rGO@MWCNT nanosystem, the FT-IR spectrum reveals additional features from MWCNTs. Peaks in the 2850–2950 cm⁻¹ range are attributed to C–H stretching vibrations, while a peak around 1580 cm⁻¹ corresponds to C=C stretching, associated with the sp² hybridized carbon atoms of MWCNTs. These peaks, along with the retained Mo–S stretching band (470–500 cm⁻¹) and characteristic peaks from rGO, confirm the successful formation of the hierarchical MoS₂@rGO@MWCNTs nanosystem. The presence of distinct peaks for each component demonstrates the effective integration of MoS₂, rGO, and MWCNTs in the composite, which is expected to enhance the material's functional properties for various applications.

3.2.5 TEM analysis. The MoS₂ NSs and MoS₂@rGO@MWCNTs nanohybrid's crystallinity and microstructure were further analyzed utilizing transmission electron microscope (TEM). Fig. 6 displays the MoS₂ NSs (a & b) and MoS₂@rGO@MWCNTs nanohybrid (c & d) TEM images using this experimental approach at two distinct magnifications. Fig. 6(a) depicts a low-magnification view, whereas Fig. 6(b) presents a high-magnification MoS₂ NSs image. The presence of grooves and ripples in the observed sample suggests that the MoS₂ NSs are both ultrathin and flexible. Fig. 6(c) and (d) illustrate the uniform dispersion of MoS₂ NSs resembling flower petals against the rGO NS background. The rGO NSs and MoS₂ NSs can be distinguished based on their thickness contrast, typically on a small nanometer scale. Fig. 6(c) and (d) show the MoS₂@rGO@MWCNTs nanohybrid structure, where the MoS₂@MWCNTs structure is prominently observed sitting atop the wider rGO NSs. Fig. 6(c) and (d) show a micrograph that displays MoS₂ wrapped around MWCNTs with rGO NSs. This observation indicates that MoS₂ NSs effectively coat the MWCNTs, forming a core–shell hybrid structure with rGO NSs. The MoS₂@MWCNTs hybrid interfaces exhibit a smooth surface, resulting in improved interfacial contact inside the nanohybrid material. Fig. 6(e) displays the high-resolution transmission electron microscopy (HR-TEM) view of the nanohybrid structure. It indicates that the lattice patterns of all the components closely align with the expected interplanar spacing of their respective nanostructures. The selected area electron diffraction (SAED) exhibits dispersive diffraction rings [Fig. 6(f)], which show low crystallization of the MoS₂@rGO@MWCNTs nanohybrid, along with broad and less intensified diffraction peaks in the XRD.

Fig. 6 TEM images of MoS₂ NS (a) and (b) and MoS₂@rGO@MWCNT nanohybrids (c) and (d) at different magnifications. Scale bar: (a) and (c) 100 and (b) and (d) 20 nm; HRTEM pictures of the MoS₂@rGO@MWCNT nanohybrid (e); and SAED pattern of the MoS₂@rGO@MWCNT nanohybrid (f).

3.3 Characterization of distinct fabricated electrodes

3.3.1 Scanning electron microscopy (SEM) analysis. The bare CC microstructure was analyzed before and after the EPD procedure, specifically for the APTES/MoS₂@rGO@MWCNTs/CC, anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC, and BSA/anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC samples that were investigated using SEM. Fig. 7(a)–(c) illustrates that the CC fiber surface lacks roughness before the EPD procedure. The CC consists of densely intertwined microfiber clusters, resulting in a somewhat coarse texture across the sample size. After a 2 min EPD treatment at 60 V, the CC surface exhibited a rough texture and was adorned with nanostructures, as depicted in Fig. 7(d)–(f). The nanohybrid APTES/MoS₂@rGO@MWCNTs distribution on each CC fiber (APTES/MoS₂@rGO@MWCNTs/CC) is evenly revealed. The unmodified CC surface morphology underwent a complete transformation, as depicted in Fig. 7(d)–(f). Fig. 7(g)–(l) shows the surface of anti-Sp17 antibody immobilization and BSA on the APTES/MoS₂@rGO@MWCNTs/CC electrode. The APTES/MoS₂@rGO@MWCNTs/CC electrodes are fully coated with a dense layer of anti-Sp17 and BSA. Following the BSA blockage procedure, a uniformly even surface was detected on the anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC substrate. The findings demonstrated the effective binding of anti-Sp17 and BSA on the APTES/MoS₂@rGO@MWCNTs/CC electrodes.

Fig. 7 SEM images of unmodified CC (a)–(c); APTES/MoS₂@rGO@MWCNTs nanohybrid-modified CC prepared at 60 V and 2 min EPD time (d)–(f); anti-Sp17 antibody-modified CC (g)–(i); and BSA modified CC (j)–(l) at three different magnifications. Scale bar: (a), (d), (g) and (j) 100 μm; (b), (e), (h) and (k) 20 μm; and (c), (f), (i) and (l) 5 μm.

3.4 Electrochemical studies

For evaluating the bare CC and modified electrode performance (CC, APTES/MoS₂/CC, APTES/MoS₂@rGO/CC, APTES/MoS₂@rGO@MWCNTs/CC, anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC, and BSA/anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC), we conducted electrochemical analyses using differential pulse voltammetry (DPV) as well as cyclic voltammetry (CV). For these measurements, an Autolab galvanostat/potentiostat electrochemical analyzer was employed with 5 mM [Fe(CN)₆]^3−/4− in 0.2 M PBS containing 0.9% NaCl as the electrolyte to facilitate Sp17 detection. Based on the results, the MoS₂@rGO@MWCNTs nanocomposite dispersion was selected for further experiments, enabling Sp17 detection at concentrations as low as 11 ng mL⁻¹ under the optimized conditions.

3.4.1 Optimum parameters for the developed biosensor. The key parameters were systematically examined to achieve optimal biosensor efficiency and sensitivity. Initially, the influence of the concentration of the APTES functionalized MoS₂@rGO@MWCNTs nanohybrid, as well as the optimal voltage and time parameters for EPD, were investigated. The detailed procedure for uniformly depositing the functionalized ternary nanosystem onto the CC electrode via EPD is given in the ESI,† Section S1 and Table S1. Subsequently, the amount of anti-Sp17 antibody required for the immunosensing system was optimized. Ten different concentrations of anti-Sp17 antibodies were employed for surface capture on the APTES/MoS₂@rGO@MWCNTs/CC nanohybrid. Fig. S1(a) (ESI†) illustrates the ΔI_p values, which increased as the anti-Sp17 antibody concentration rose from 10 to 70 μg mL⁻¹, indicating enhanced binding due to increased active groups for stronger bonds. Beyond 80 μg mL⁻¹ antibody immobilization, the ΔI_p values did not further increase, leading to the selection of 60 μg mL⁻¹ anti-Sp17 antibodies for constructing the immunosensing system. Moreover, the incubation period emerged as a critical factor influencing the interaction of anti-Sp17 antibodies with the target Sp17 antigen and, consequently, the sensitivity of the detection strategy. The incubation period for anti-Sp17 antibody immobilization on the APTES/MoS₂@rGO@MWCNTs/CC electrode was studied over 1 to 10 h. Fig. S1(b) (ESI†) demonstrates that ΔI_p increased with the incubation period of up to 6 h, stabilizing with further increments. Hence, 6 h of incubation was deemed optimal. Furthermore, optimizing the BSA concentration and incubation time is vital for achieving maximum biosensor performance, balancing surface coverage needs with avoiding non-specific binding and saturation effects. Fig. S1(c) (ESI†) depicts the variations in DPV peak current of the BSA/anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC immunoelectrode with BSA concentration across a range of concentrations from 0.1% to 4%. Peak current showed a noticeable increase within the 0.1% to 2% concentration range, reaching saturation beyond this concentration. Thus, a 2% BSA concentration was optimal for subsequent experiments. In addition, six different incubation times (0–180 min) were tested to assess their impact on binding efficiency to the sensor surface and subsequent immobilization of target biomolecules. An increase in ΔI_p value was observed with increasing BSA incubation time up to 120 min, beyond which saturation occurred [Fig. S1(d), ESI†]. Therefore, 120 min was selected as the optimal BSA incubation time. These optimization experiments were repeated three times for reliability. Table 1 outlines the optimal results corresponding to each variable under investigation.

Table 1 Different experimental optimized parameters

Variable	Tested	Selected
EPD parameter of APTES/MoS2@rGO@MWCNTs/CC deposition (concentration, voltage & time)	Concentration – 5, 10, 50, 100, 150, 200, 250, & 300 μL	200 μL, 40 V, and 90 s
	Voltage – 30, 40, 50, 60, & 70
	Time – 60, 90 & 120 s
Anti-Sp17 conc.	10, 20, 30, 40, 50, 60, 70, 80, 90, & 100 μg mL^−1	60 μg mL^−1
Anti-Sp17 incubation time	0, 1, 2, 3, 4, 5, 6, 7, 8, 9, & 10 h	6 h
BSA concentration	0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, & 4.0%	2%
BSA incubation time	0, 30, 60, 90, 120, 150, & 180 min	120 min
pH	6.0, 6.5, 7.0, 7.5, & 8.0	7.0
Response time	0, 1, 2, 3, 4, 5, 6, 7, 8, 9, & 10 min	5 min

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The impact of pH on the BSA/anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC electrode has been examined using DPV at a 50 mV s⁻¹ scan rate in a PBS solution comprising redox species. The current response exhibits its highest magnitude at a neutral pH level of 7.0, as shown in Fig. 8(a). This could be attributed to biological components, such as antibodies, enzymes, amino acids, antigens, etc., in their native state, which exhibit significant activity at a pH of 7.0. Yet, in a basic or acidic environment, antibodies undergo denaturation owing to the interaction between H⁺/OH⁻ with the antibodies’ amino acid sequence.⁵² Therefore, all electrochemical investigations have been carried out utilizing PBS with a pH of 7.0 and a redox probe.

Fig. 8 pH analysis (a); CV (b) and DPV (c) spectra of the proposed immunosensor during the stepwise modification of CC; CV of APTES/MoS₂@rGO@MWCNTs/CC (d) and BSA/anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC (e) electrodes at increasing scan rate (10–100 mV s⁻¹); I_pa and I_pc against square root plot of scan rate [d(i) and e(i)]; and ΔE_p against square root plot of scan rate [d(ii) and e(ii)].

The stepwise fabrication and electrochemical behavior of the immunosensor were examined using CV with a 50 mV s⁻¹ scan rate and potential window of −0.8 V to +0.8 V. DPV was also performed in 0.2 M PBS with a neutral pH, including a redox coupler.

3.4.2 Electrochemical characterization of the fabricated electrodes. CV curves for the unmodified CC were utilized as a baseline for assessing the modified electrode performance, including APTES/MoS₂/CC, APTES/MoS₂@rGO/CC, APTES/MoS₂@rGO@MWCNTs/CC, anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC, and BSA/anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC [Fig. 8(b)]. On comparison analysis, improvement in electrochemical performance was seen following modifications with MoS₂, rGO, MWCNTs, and their nanocomposite combinations were determined, each showing an enhanced current response, shifts in redox potentials, increased electrochemical stability, and more efficient charge-transfer kinetics. The CV results display distinct redox peaks for each modified electrode, demonstrating both shape and intensity consistent with the reversible behavior of the redox species used in this work.

We evaluated all the electrodes by assessing the potential difference between anodic peaks and peak heights. The outcomes depicted that every electrode arrangement had responses in our CV analysis. The bare CC showed a scarcely defined and widely defined pair of peaks using CV, which had an anodic peak current (I_pa) of 1167.29 μA (curve i) and a peak potential separation (ΔE) of −0.224 V. These values acted as a reference to compare the modified electrodes and indicated a more sluggish and diverse electron transport pathway. Incorporating APTES/MoS₂ onto the CC resulted in a peak current increase to 1306.45 μA (curve ii), demonstrating enhanced electrochemical activity. Additionally, the ΔE decreased to −0.480 V, indicating a shift in redox behavior that suggests that APTES/MoS₂ promotes charge transfer and alters the electrode's electronic properties. Further improvement was observed with the APTES/MoS₂@rGO modified electrode, showing a peak current of 1485.59 μA (curve iii) and ΔE of −397 V, which indicates even greater electrochemical activity, likely due to the properties of the APTES/MoS₂@rGO material.⁵³ The highest enhancement was seen with the APTES/MoS₂@rGO@MWCNTs nanocomposite, reaching a peak current of 1800.84 μA (curve iv) and a ΔE of −0.305 V. The combined effect of MoS₂, rGO, and MWCNTs in this composite provides a larger surface area and facilitates electron transfer, significantly enhancing electrochemical performance.⁵⁴ Thus, based on peak current and peak potential separation, the APTES/MoS₂@rGO@MWCNTs nanocomposite was chosen as the optimal material for Sp17 protein detection. Nevertheless, following the anti-Sp17 antibody immobilization over the APTES/MoS₂@rGO@MWCNTs/CC electrode, an alteration of peak current (ΔI_pc) of 2153.93 μA; curve (v) and ΔE of −331 improve, and the peak potential moves towards a lower value, suggesting an effortless movement of electrons to the electrode surface. The APTES/MoS₂@rGO@MWCNTs can serve as a bridge on the electrode surface, effectively reducing the electron tunneling distance between the electrode and antibodies. This leads to an increased current. In addition, the electrostatic interaction between the unoccupied region of the antibodies (–NH₂ terminal) and the redox probe can lead to rapid electron transport toward the immunoelectrode.¹⁵ Following the immobilization of BSA, the change in I_pc drops to a value of 1922.91 μA and ΔE of −0.288 V, as seen by curve vi. The reason for this is that the uncovered APTES/MoS₂@rGO@MWCNTs locations of the anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC immunoelectrode have non-specific BSA loading. Following the application of BSA, the peak potential shifts towards higher values owing to the insulating properties exhibited by the molecules of BSA.⁵⁵ In addition, DPV was determined using a 5 mV step potential within the potential range spanning from −0.8 to +0.8 V at intervals of 0.5 ms, having a scan rate of 50 mV s⁻¹ [Fig. 8(c)]. The DPV analyses yielded findings that were in agreement with the CV measurement findings, indicating the effective development of a BSA/anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC biosensor.

The transferability of electrons at the bare CC, APTES/MoS₂/CC, APTES/MoS₂@rGO/CC, APTES/MoS₂@rGO@MWCNTs/CC, anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC, and BSA/anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC are studied in 0.2 M PBS (0.9% NaCl) utilizing CV at 50 mV s⁻¹ scan rate, and the potential range spanning from −0.8 to +0.8 V versus Ag/AgCl was employed to measure them, as shown in Fig. 8(b). The cathodic as well as anodic peak potentials (E_pc and E_pa), reduction and oxidation peak current densities (J_pa and J_pc), and the potential difference (ΔE_p) are evaluated in Table 2. The anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC electrode exhibited reduced E_p and more pronounced redox peak currents compared to other unmodified and modified electrodes, as shown in Table 2. This can be attributed to the favorable electrostatic interactions among the [Fe(CN)₆]^3−/4− and APTES/MoS₂@rGO@MWCNTs surface.

Table 2 CV parameters of various modified electrodes

S. no.	Electrodes	E pa (V)	E pc (V)	J pa (μA cm^−2)	J pc (μA cm^−2)	ΔEp (mV)
1	Bare CC	0.030	0.254	−1133.42	1167.29	−224
2	APTES/MoS2/CC	−0.072	0.408	−1264.34	1306.45	−480
3	APTES/MoS2@rGO/CC	−0.035	0.362	−1463.01	1485.59	−397
4	APTES/MoS2@rGO@MWCNTs/CC	0.003	0.308	−1750.18	1800.84	−305
5	Anti-Sp17/APTES/MoS2@rGO@MWCNTs/CC	−0.013	0.318	−2059.93	2153.93	−331
6	BSA/anti-Sp17/APTES/MoS2@rGO@MWCNTs/CC	0.003	0.291	−1863.40	1922.91	−288

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3.4.3 Scan rate studies. The electrochemical behavior of the APTES/MoS₂@rGO@MWCNTs/CC, as well as BSA/anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC electrodes, is investigated by changing the scan rate (10 to 100 mV s⁻¹). The CV results for the two electrodes are shown in Fig. 8(d) and (e). The values of I_pc and I_pa peak current vary linearly concerning the square root of the scan rate. These values, as shown in Fig. 8d(i) and e(i), do not cross the origin and are determined by eqn (1)–(4). This indicates that the electrochemical procedure is diffusion-controlled.^56,57

I_{pa(APTES/MoS2@rGO@MWCNTs/CC)} (μA) = [262.97 μA (mV s⁻¹)^1/2 × sqrt scan rate (mV s⁻¹)^1/2] + [−105.47 μA], R² = 0.999 (1)

I_{pa(BSA/anti-Sp17/APTES/MoS2@rGO@MWCNTs/CC)} (μA) = [268.88 μA (mV s⁻¹)^1/2 × sqrt scan rate (mV s⁻¹)^1/2] + [−45.10 μA], R² = 0.999 (2)

I_{pc(APTES/MoS2@rGO@MWCNTs/CC)} (μA) = −[274.56 μA (mV s⁻¹)^1/2 × sqrt scan rate (mV s⁻¹)^1/2] + [166.25 μA], R² = 1 (3)

I_{pc(BSA/anti-Sp17/APTES/MoS2@rGO@MWCNTs/CC)} (μA) = −[264.01 μA (mV s⁻¹)^1/2 × sqrt scan rate (mV s⁻¹)^1/2] + [47.76 ± 13.61 μA], R² = 0.999 (4)

Also, the increasing scan rate affects the anodic besides cathodic peak potentials (E_pa and E_pc). On proceeding from a larger to smaller timescale, the positive and negative potential shifts of E_pa and E_pc are visible [Fig. 8d(ii) and e(ii)], leading to increased separation between peak potentials (ΔE_p). This separation potential (ΔE_p = E_pa − E_pc) also follows a linear trend with square root scan rate as given by eqn (5) and (6), and the values are more than the value of , suggesting quasi-reversible interfacial electron transfer kinetics.^58,59

ΔE_{p(APTES/MoS2@rGO@MWCNTs/CC)} (mV) = [0.037 ± 0.89 mV (mV s⁻¹)^1/2 × sqrt scan rate (mV s⁻¹)^1/2] + [0.027 ± 9.16 mV], R² = 0.999 (5)

ΔE_{p(BSA/anti-Sp17/APTES/MoS2@rGO@MWCNTs/CC)} (mV) = [0.035 mV (mV s⁻¹)^1/2 × sqrt scan rate (mV s⁻¹)^1/2] + [0.0632 ± 9.16 mV], R² = 0.999 (6)

The kinetic interface parameters of the BSA/anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC and APTES/MoS₂@rGO@MWCNTs/CC electrodes were evaluated to investigate electron transfer dynamics. Key parameters like the diffusion coefficient (D), surface concentration of redox species (I*), standard heterogeneous electron transfer rate constant (K_s), and effective surface area (A_e) were calculated. Detailed calculations for these parameters are given in the ESI† (Section S2), with the resulting kinetic data for both electrodes summarized in Table S2 (ESI†).

3.4.4 Electrochemical response studies. Subsequently, to track the duration needed for the whole interaction between the BSA/anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC immunoelectrode and Sp17, and to verify the definite antibody-antigen immunocomplex formation, response time experiments were carried out. These experiments involved monitoring periodic variations in I_pa after the Sp17 antigen addition. It is noted that the I_pa gradually falls until it reaches a maximum of 5 min. Afterward, the curve level stops and a consistent response is achieved, as depicted in Fig. 9(a). Thus, we have selected the sample incubation time as 5 min, during which the antigen–antibody binding reaction reaches the steady state.

Fig. 9 Response time study (a); sensing response of BSA/anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC versus Sp17 concentrations (0.01–11 ng mL⁻¹) through DPV techniques (b); enlarged view of the electrochemical response study (c); linear graph showing peak current against Sp17 concentration (d); Hanes–Wolf plot (e); control studies showing electrochemical response of APTES/MoS₂@rGO@MWCNTs/CC versus increasing Sp17 concentrations (0.01–11 ng mL⁻¹) (f); interferent analysis employing different analytes that exist in human serum along with Sp17 (g); reproducibility study showing anodic peak current values of different immunoelectrodes (BSA/anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC) developed under similar experimental parameters (h); outcomes of repeatability study (i); reusability (j); and storage stability (k) of the immunosensor.

Subsequently, the electrochemical reaction of the constructed immunoelectrode was examined against Sp17 utilizing the DPV approach, with concentrations extending from 0.01 to 11 ng mL⁻¹. The DPV technique enhanced the sensitivity, providing well-defined peaks that exhibit accuracy in both shape and position. The clear correlation between current values and analyte concentration underscores the method's reliability, streamlining the calibration process and reinforcing its quantification suitability. This linear relation ensures that the measured current is directly proportional to concentration, enabling straightforward and accurate analyte detection. We observed through experimentation that the DPV current diminishes progressively as the concentration of Sp17 increases [Fig. 9(b) and (c)]. The decrease in peak current is ascribed to the expanded size of the electrode surface following the Sp17 attachment with the anti-Sp17 antibody on the electrode surface. A bulkier surface area would hinder the movement of electrons, leading to a decrease in the peak current response.¹⁵ In addition, the MoS₂@rGO@MWCNTs nanohybrid has transfer pathways that provide the movement of electrons between the electrode as well as an electrolyte that acts as both an electron donor and acceptor, improving the process of transfer of electrons and increasing the electrochemical signals. Hence, it achieves a broad linear range of 0.01–11 ng mL⁻¹, along with a quantification limit (LOQ) of 0.79 ng mL⁻¹ and a detection limit (LOD) of 0.23 ng mL⁻¹.⁵³ The evaluation of reproducibility was performed 3 times. The inclusion of error bars was based on the standard error of 3 trial tests. With this remarkable LOD, the biosensor could identify as little as 1.12 × 10⁻⁴ Sp17 molecules, enabling the detection of extremely low Sp17 concentrations (the detailed calculation has been provided below).

3.4.4.1 Calculation of Sp17 molecules at a detection limit of 0.23 ng mL⁻¹. Sp17 molecular weight (M_w) = 24.5 kDa or 24.5 × 10³ g mol⁻¹

Sp17 solution’ concentration (strength) at detection limit = 0.23 ng mL⁻¹ or 2.3 × 10⁻⁷ g L⁻¹

So, the number of moles in 10⁶ μL/1 L of solution = 5.6 × 10¹² molecules.

Therefore, the number of moles in 20 μL of solution

Hence, 20 μL of 0.23 ng mL⁻¹ solution comprises 1.12 × 10⁻⁴ molecules of Sp17.

The immunosensor LOD and LOQ were determined using eqn (7) and (8), respectively.⁶⁰

where s depicts standard deviation (SD) [SD = S.E. × √N], m and S.E. signify the slope's standard error and intercept, respectively, and N denotes the number of samples.

A calibration plot was made by charting the variations in I_pa of the BSA/anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC immunoelectrode versus the Sp17 protein concentration in the 0.01–11 ng mL⁻¹ detection range [Fig. 9(d)]. The resultant graph followed a linear equation (eqn (9)).

ΔI_p = [−2.493 (μA mL ng⁻¹) × Sp17 conc. (ng mL⁻¹)] + 397.32 μA, R² = 0.97 (9)

Furthermore, the 3D structured MoS₂@rGO@MWCNTs ternary nanosystem functions as a remarkably efficient substrate because of its strong non-covalent attachment to biomolecules. This enables binding of a significant amount of anti-Sp17 antibodies, leading to an outstanding 9.97 μA ng mL⁻¹ cm⁻² sensitivity when employing DPV approaches. The biosensor's increased sensitivity and widened detection range can be attributed to the existence of an extremely sensitive probe composed of oxygen-containing groups and an elevated quantity of surface imperfections, specifically implanted over the MoS₂ NSs and rGO NSs, leading to an amplified electrochemically active surface area. Moreover, the combined attributes of MoS₂, rGO, and MWCNTs have been noted for their broad surface pores and remarkable electrical conductivity, resulting in the highly efficient transmission of electrons in an electrolytic probe. Therefore, the MoS₂@rGO@MWCNTs biosensor that was formed exhibits outstanding biosensing properties for accurately detecting the Sp17 biomarker. Table 3 displays a comparative analysis of analytical characteristics for detecting the Sp17 biomarker employing multiple biosensors.

Table 3 Depiction of biosensing parameters for comparison from earlier reported biosensors for SP17 determination

Immunoelectrodes	Technique	Sensitivity (μA mL^−1 ng^−1 cm^−2)	Linear detection range (ng mL^−1)	Detection limit (ng mL^−1)	Response time (min)	Regeneration	Shelf-life (week)
BSA/anti-SP-17/APTMS/ITO	DPV	0.013	0.1–5	0.7	30	5 (90%)	5
BSA/anti-SP17/GPTMS@SAMs/ITO	CV	0.047	0.1–6	0.4	15	5 (95%)	8
	DPV	0.024	0.05–5.5	0.142
BSA/anti-Sp17/APTES/nMoS2 NS@rGO/ITO	DPV	12.21	0.05–8	0.13	20	6	7
	EIS	0.48	1–8	0.23
BSA/anti-Sp17/APTES/nMoS 2 NS@rGO@MWCNTs/CC	DPV	9.97	0.01–11	0.23	5	7 (88.3)	10 (89%)

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3.4.4.2 Detection mechanism based on Fermi level fluctuations induced by charge transfer. The adsorption of chemical species on semiconductor surfaces can be analyzed in terms of two main aids to adsorption energy: charge transfer and bond formation. The direction of the Fermi level shift inside the bandgap is determined by charge transfer, whether from the species to the semiconductor or the reverse. The adsorption energy is devoid of any bulk doping effects, and the Fermi energy in semiconductors with many defect states is close to the Fermi level, where the Fermi level is “pinned” to the surface.

For surfaces where the Fermi level is not pinned, as in our case, the adsorption energy is affected by changes in bulk Fermi energy through doping or charge transfer. A scheme showing the charge transfer-dependent Fermi level shift for both n-type and p-type semiconductors is shown in Scheme 2, with specific scenarios as follows:

Scheme 2 Illustrative diagram depicting the general detection mechanism – in this setup (a–d), either adsorption of a donor or acceptor molecule onto the MoS₂@rGO@MWCNT surface-based sensor electrode. Here, ‘M’ represents the metal contact, while ‘S’ denotes the p-type semiconductor properties of the MoS₂@rGO@MWCNTs.

(a) In thermal equilibrium (no sensing), the Fermi levels of the metal (E_FM) and the MoS₂@rGO@MWCNTs composite (E_FS) align to form a single Fermi level in the metal-semiconductor system.

(b) During sensing, the Fermi level of MoS₂@rGO@MWCNTs (E_FS) shifts up or down depending on charge transfer direction from the MoS₂@rGO@MWCNTs to functional groups or vice versa. If the accumulation of holes in the valence band occurs by accepting charge from a target species, EFS moves toward the valence band, increasing the p-type conductivity.

(c) When donor molecules attach to the p-type MoS₂@rGO@MWCNTs surface, electrons accumulate in the conduction band, shifting EFS towards the conduction band. This corresponds to hole depletion in the valence band, reducing conductivity and increasing resistance.

(d) Prolonged exposure to donor/acceptor molecules can create a high amount of defect states near E_FS, redistributing charge carriers across states above and below the Fermi level. This “Fermi level pinning effect” prevents further Fermi level shifts, resulting in sensor response saturation and minimal conductivity changes.

The described mechanisms in Scheme 2(a)–(d) are in the current sensing study. Our proposed sensor operates by detecting Fermi level fluctuations owing to the adsorption of either acceptor or donor molecules on the p-type MoS₂@rGO@MWCNTs, with high sensitivity to Sp17 concentration, achieving a detection limit as low as 0.23 ng mL⁻¹. A comparative analysis in Table 3 showcases the Sp17 sensing performance against existing biosensors. The MoS₂@rGO@MWCNTs nanosystem demonstrated superior performance in sensitivity and accuracy compared to unmodified CC and CC modified with either MoS₂ or MoS₂@rGO. The correlation coefficients (R²), regression equations, slopes, and standard errors for these materials are shown in eqn (9). The strong R² values support the effectiveness of the MoS₂@rGO@MWCNTs nanosystem as a promising material for electrochemical sensing uses, offering enhanced accuracy and sensitivity, a notable electroanalytical chemistry advancement. Additionally, we compared the performance characteristics of MoS₂@rGO@MWCNTs/CC with previously reported Sp17 biosensors (Table 3).

3.4.4.3 Hanes–Wolf plot for dissociation and association constant values. The association constant (K_a) value was determined by generating a Hanes–Wolf plot correlating Sp17 concentration with the ratio of the immunoelectrode's current to Sp17 concentration, yielding a value of 370.37 ng mL⁻¹ [Fig. 9(e)]. The K_a value can vary depending on factors such as the attachment sites of biomolecules and how antibodies bind to the electrode's surface, resulting in different conformational changes in antibody arrangement. The enhanced K_a value, attributed to the positive conformation of anti-Sp17 and higher electrode surface loading, indicates a strong affinity of the BSA/anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC immunoelectrode towards Sp17. The dissociation constant (K_d) value, estimated at −758.59 ng mL⁻¹, further underscores Sp17's specific affinity. The K_a value was estimated utilizing Hanes–Wolf linear graphs derived from the slope inverse, where the product of K_a and intercept equates to K_d.^15,61

3.4.5. Control, interferent, reproducibility, and repeatability studies. To check the variable current that resulted from the interaction between Sp17 and the BSA/anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC immunoelectrode, a control experiment was performed utilizing the APTES/MoS₂@rGO@MWCNTs/CC electrode. Subsequently, the electrochemical response was measured by gradually adding higher quantities of Sp17. Fig. 9(f) demonstrates no significant variation in the anodic peak current. Thus, it can be deduced that the formation of immunocomplexes is solely accountable for the changes in peak current values observed in electrochemical response studies.

To further assess the specificity of the developed platform towards Sp17, various interfering agents (glucose, urea, oxalic acid, ascorbic acid, uric acid, NaCl, IL-8, CYFRA-21-1) present in human serum were introduced one after the other to measure the electrochemical response of the BSA/anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC immunoelectrode. The selection of interfering species for the selectivity assays was based on the typical composition of human serum, where electroactive and structurally diverse molecules, as mentioned above, are frequently present. These analytes were specifically chosen to simulate realistic physiological conditions and to assess the biosensor's specificity for Sp17 amidst potentially confounding species. As shown in Fig. 9(g), there is a noticeable alteration in the current only after the introduction of Sp17. Subsequently, no notable variation is observed when interferents are added. Therefore, the developed biosensing platform demonstrated a solid ability to detect Sp17 specifically. The results indicate that the materials and methods are reliable, making this approach well-suited for applications requiring selective detection of Sp17.

Next, to ensure the immunosensor's reproducibility, 10 distinct electrodes (BSA/anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC) were developed using comparable experimental conditions. The electrochemical response was then measured using the DPV approach. The anodic current peak values of several electrodes are depicted as a bar plot in Fig. 9(h). The study revealed that the average %RSD of the peak current for various immunoelectrodes was around 4.125%. This indicates that the fabricated biosensing electrode has a high level of reproducibility. Additionally, the repeatability of the constructed immunoelectrode was calculated using the DPV technique. This required taking 6 successive readings for a particular concentration of Sp17 (11 ng mL⁻¹), as shown in Fig. 9(i). The repeatability test revealed a minor deviation in current, with a satisfactory %RSD of 0.43%. These results affirm the capability of the immunoelectrode to deliver consistent and reproducible outcomes with sufficient precision.

3.4.6 Reusability and storage stability of the immunosensor. While the CC electrode is inexpensive, the ability to reuse the suggested immunosensor is a significant benefit for practical investigation. The BSA/anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC electrode was made carefully under the determined experimental conditions to conduct the reusability test. Next, the electrode was exposed to the Sp17 antigen using the DPV technique, and the electrode signal was recorded. Following the DPV evaluation, the electrode was submerged in a 0.1% HCl solution for 5 min to eliminate any specific contact between the antigen and the antibody. The DPV electrode signal was tested again after immersing it in an acidic solution. The process was carried out until the immunosensor failed to exhibit a response. According to Fig. 9(j), the immunosensor did not respond significantly after the 7th acid treatment. Furthermore, the acid solution caused harm to the immunosensor surface. The immunosensor demonstrated a retention rate of 96.66% and 88.30% for its initial use until the 6th and 7th acid treatment, respectively [Fig. 9(j)].

Additionally, the biosensors' storage stability is a crucial characteristic for their clinical application and was assessed by measuring the DPV of the BSA/anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC electrode over one week. This technique was performed weekly. The DPV response maintained 98% of its peak current until the 8th week, gradually decreasing to 89% and 85% by the 9th and 10th weeks, respectively [Fig. 9(k)]. The result indicated that the constructed biosensor had remarkable storage stability for 8 weeks.

3.5 Real sample analysis

The ELISA method was employed to measure the quantity of Sp17 in serum samples obtained from 10 cancer patients. The experiment was conducted in triplicate for both serum samples and standard solutions. The double-antibody sandwich ELISA kit (CUSABIO; Catalog No. CSB-EL022451HU) is carried out in 96-well microtiter plates precoated with anti-Sp17 antibodies. Following the assay protocol, a colorimetric reaction is induced, and the absorbance is measured at 450 nm using an ELISA plate reader. The accuracy of the biosensor for assessing the electrochemical response of blood specimens utilizing DPV has been tested using a range of Sp17 concentrations in serum samples collected by ELISA (Table 4). It is evident that there is a decent association between the standard Sp17 concentration and DPV current response of the manufactured immunoelectrode in the presence of (a) concentrations of Sp17 in serum samples as assessed by ELISA as well as Sp17 standard concentration (Table 4 and Fig. 10). The results demonstrate a concise and consistent average %RSD of around 0.31% between the current response of serum samples and standard solutions. This indicates that the developed biosensor exhibits a high level of accuracy for its application in detecting cancer biomarkers. The sensing parameters of the developed immunosensors and earlier reported biosensors are described in Table 3. In summary, the results presented in Table 4 indicate that the analytical techniques are reliable, showing high precision and recovery in both standard and serum samples, especially at higher concentrations. The developed sensor demonstrated strong sensitivity for both sample types, as evidenced by its low LODs and LOQs.

Table 4 Percentage relative standard deviation obtained using the BSA/anti-Sp17/APTES/MoS₂@rGO@MWCNTs/CC electrode with standard and patient samples through DPV

Patient sample number	Conc. of Sp17 quantify using ELISA (in pg mL^−1)	Peak current (μA) in standard samples	Peak current (μA) in serum samples	% RSD	Recovery (%)
1	58.94	400.67	399.13	0.27	99.61
2	206.74	397.92	395.92	0.35	99.49
3	336.97	397.45	395.45	0.35	99.49
4	25.26	402.62	401.45	0.20	99.70
5	52.21	401.06	399.59	0.26	99.63
6	4480.41	383.63	381.55	0.38	99.45
7	450.50	397.04	395.04	0.35	99.49
8	3677.62	385.10	383.39	0.31	99.55
9	275.2	397.67	395.67	0.35	99.49
10	523.96	396.77	394.77	0.35	99.49
	Average %RSD			0.317	99.539

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Fig. 10 Bar graph illustrating the current comparison obtained using DPV of cancer patients and standard samples.

4. Conclusions

In this study, we developed a novel 3D flexible electrochemical biosensor using a MoS₂@rGO@MWCNTs ternary nanohybrid for the sensitive detection of the cancer biomarker Sp17. This innovative platform is one of the very few reported to date for Sp17 detection, with only three other systems known. Our approach involved the integration of anti-Sp17 antibodies onto a conductive CC substrate using electrophoretic deposition, enhanced with MoS₂@rGO@MWCNTs nanomaterials. The nanohybrid structure, characterized by XPS, XRD, Raman, FT-IR, TEM, and electrochemical methods, showed excellent physical and electrochemical properties. The incorporation of MWCNTs improved the dispersion of the MoS₂ and rGO, preventing agglomeration and providing abundant sites for antibody immobilization. The resulting biosensor exhibited exceptional performance, including a wide linear detection range (0.01–11 ng mL⁻¹), high sensitivity (9.97 μA mL ng⁻¹ cm⁻²), and a very low detection limit (0.23 ng mL⁻¹), capable of detecting as few as 1.12 × 10⁻⁴ Sp17 molecules. Other key advantages include LOQ (0.79 ng mL⁻¹), rapid response (within 5 min), high stability (up to 10 weeks), strong regeneration (up to 6 cycles), good reproducibility (RSD = 0.43%), and good selectivity under ambient conditions. When tested with real human serum samples, the biosensor delivered reliable results, which were in strong agreement with standard ELISA tests, validating its potential for clinical applications. Importantly, this biosensor is not only cost-effective and scalable but also adaptable for point-of-care (POC) diagnostics. The synergistic effects of MoS₂, rGO, and MWCNTs contribute significantly to its outstanding sensing capabilities. Overall, the proposed MoS₂@rGO@MWCNTs based sensor holds great promise for future use in cancer diagnostics, wearable biosensing devices, and broader applications in disease detection.

Live subject statement

All participants involved in this study provided informed consent before their involvement.

Author contributions

Amit K. Yadav: investigation, conceptualization, formal analysis, validation, writing – original draft, writing – review & editing. Damini Verma: formal analysis, writing – original draft, formal analysis, writing – review & editing. Pratima R. Solanki: writing – review & editing, validation, supervision, funding acquisition.

Conflicts of interest

The authors confirm that there are no financial or personal conflicts of interest that could have influenced the results or interpretation of this study.

Data availability

The datasets generated or analyzed during the study are accessible from the corresponding author upon reasonable request.

Acknowledgements

The authors sincerely acknowledge the Advanced Instrumentation Research Facility (AIRF) at Jawaharlal Nehru University (JNU) for access to the essential characterization instruments. Partima Solanki acknowledges financial support from the Biomedical Device and Technology Development (BDTD) program, the Department of Science and Technology (DST), and the Indian Council of Medical Research (ICMR), both based in New Delhi. Amit K. Yadav extends gratitude for the Prime Minister Research Fellowship funding awarded by the Ministry of Education, Government of India.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5tb00741k

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