Ultrasensitive electrochemical biosensor based on MXene quantum dots for prostate cancer biomarker detection

Prostate cancer is the second most common cancer among men, accounting for approximately 7% of cancer-related fatalities worldwide. Nearly 50% of men over the age of 70 will eventually develop PSA¹. Prostate-specific antigen (PSA) is widely recognized as a biomarker for the clinical assessment of prostate cancer². Thus, the ability to detect PSA in a straightforward, rapid, and precise manner is crucial for the early diagnosis of prostate cancer and for preventing disease recurrence. Normal PSA levels in the bloodstream are typically < 4.0 ng mL^-1, while levels between 4.0 and 10.0 ng mL^{− 1} are considered a diagnostic “gray zone”³. Thus, accurate and rapid detection of PSA is essential for timely diagnosis and disease management.

Most PSA tests are conducted in advanced laboratories using conventional methods, leading to higher administrative and healthcare costs. Various analytical techniques such as colorimetric assays ⁴, radioimmunoassay⁵, surface plasmon resonance⁶, fluorescence immunoassay⁷, surface enhanced Raman spectroscopy⁸, chemoluminescence immunoassays⁹, and electro-phoretic immunoassay¹⁰ have been employed for PSA detection. Electrochemical immunoassays have garnered significant attention due to their high sensitivity, cost-effectiveness, and ability to detect biomarkers with precision. The sensitivity of such assays can be further enhanced through antibody quantification and the incorporation of nanoparticles to amplify signals. Incorporating engineered nanomaterials-particularly those with high conductivity and large surface area-has become a fundamental strategy for enhancing signal transduction and biorecognition. Diverse nanomaterials such as metal-organic frameworks (MOFs)¹¹, Graphene oxide/Cu–MOF¹², MWCNTs-COOH/Fc-COOH@COAl-LDH nanocomposite¹³, carbon quantum dots¹⁴, silica nanoparticles¹⁵, MXenes carbon nanotube (CNT)@AuNPs¹⁶, Fe₃O₄ ionic liquid¹⁷, Polyaniline-Loaded MXene and Gold-Decorated β-Cyclodextrin¹⁸ have been extensively employed in biosensor fabrication to immobilize antibodies.

MXenes are a class of two-dimensional transition metal carbides, nitrides, or carbonitrides with a unique combination of properties, making them versatile materials. They are derived from layered ternary carbide, nitride, or carbonitride precursors, known as MAX phases, by selectively etching the “A” element (often aluminum) to produce a 2D layered structure. MXenes feature surfaces rich in functional groups like hydroxyl and fluorine terminations, facilitating easy surface modifications and compatibility with other materials. Certain MXenes, such as titanium carbide (Ti₃C₂Tx), exhibit exceptional photothermal properties, making them valuable in applications like nanocomposites for cancer treatment¹⁹.

When MXenes are reduced to nanoscale dimensions (e.g., below 10 nm), they transform into MXene quantum dots, which retain many of the parent material’s unique benefits while gaining enhanced properties such as increased specific surface area²⁰, improved biocompatibility²¹, and easier doping or functionalization²². MXene quantum dots also exhibit significantly lower cytotoxicity compared to their larger MXene counterparts, making them more suitable for detecting chemical substances in biological systems²³. These advantages position MXene quantum dots as promising materials for sensing applications, enabling the development of high-performance sensors²⁴.

However, the use of MXene quantum dots for biomarker detection via electrochemical techniques is still in its early stages²⁵. The incorporation of PANI as a conductive polymer matrix improves dispersion and provides additional electroactive sites, while Au-NPs contribute to antibody conjugation efficiency and signal amplification²⁶. Electrode fabrication via hydrothermal synthesis, drop-casting, and nanocomposite layering allows controlled optimization of morphological features, active surface area, and electrochemical accessibility. These factors jointly influence key biosensor metrics such as reproducibility, limit of detection, and dynamic response range^27,28. The function of electrochemical biosensors is dominated by the incorporation of materials that are advanced both structurally and functionally at the electrode interface. Specifically, the synergistic utilization and the rational combination of MXene quantum dots, conductive polymers, and noble metal nanoparticles determine the sensor’s operational sensitivity, electron transfer efficiency, and biofunctionality. The presented work takes advantage of the composite of PANI@Ti₃C₂ MXene quantum dots with AuNPs as the signal capture element, merged with V₂C MXene quantum dots as signal tags, to accomplish a sandwich-type immunosensor for ultra-trace PSA detection.

Materials and reagents

Mouse anti-human monoclonal antibody (Ab₁), Prostate specific antigen (PSA), Mouse anti-human monoclonal antibody (Ab₂), Alpha-Fetoprotein (AFP), Human Immunoglobulin G (IgG), carcinoembryonic antigen (CEA), Streptavidin (STR), Glutaraldehyde (GA), Bovine serum albumin (BSA), Phosphate buffer (0.1 mol L^− 1, pH 7.0), Human blood serum (healthy man), Ultrapure water, Hydrogen peroxide (H₂O₂), Tetrapotassium hexacyanoferrate trihydrate (K₄[Fe(CN)₆])³⁻, Sodium dihydrogen phosphate dehydrate (NaH₂PO₄.2H₂O), Disodium hydrogen phosphate dehydrate (Na₂HPO₄.2H₂O), Titanium aluminium carbide (Ti₃AlC₂) powder, Vanadium aluminium carbide (V₂AlC) powder, Hydrofluoric acid (HF), Hydrogen tetrachloroaurate (III) hydrate (HAuCl₄·3H₂O), dimethyl sulfoxide (DMSO) and Polyaniline (PANI) powder.

Apparatus

All electrochemical experiments, including cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS), were conducted using a Metrohm Autolab system (Autolab PGSTAT 302-N, Netherlands) controlled by Nova software (version 2.1.8). A glassy carbon electrode (GCE) was employed as the working electrode, with a Ag/AgCl electrode saturated with potassium chloride serving as the reference electrode, and a platinum wire electrode as the counter electrode.

The morphological and compositional analyses were performed using a field-emission scanning electron microscope (FE-SEM, MIRA3, Czech Republic) and an energy-dispersive X-ray analyzer (EDX, OXFORD, England). Transmission electron microscopy (TEM) was carried out using a Zeiss EM10 microscope (Germany). Additional characterization techniques included Fourier transform infrared spectroscopy (FT-IR, PerkinElmer Spectrum 2, USA), and X-ray diffraction spectroscopy (XRD, Rigaku Ultima IV, Japan).

Approval of ethics and consent for participation

This study was conducted in full compliance with all applicable regulations and received approval from the local ethics committee of Tehran University of Medical Sciences (Approval No. IR.TUMS.TIPS.REC.1402.134). All participants were informed about the study and provided written informed consent, which was successfully obtained prior to their participation.

Synthesis of the titanium MXene quantum dots

A total of 2.0 g of Ti₃AlC₂ MAX powder was dispersed in 240 mL of hydrofluoric acid (HF, 40 wt%) and stirred for 64 h. The resulting mixture was washed with deionized (DI) water until it reached a neutral pH. The mixture was then centrifuged and repeatedly washed until the pH stabilized at approximately 6.0, after which it was dried at 60 °C. Subsequently, 0.5 g of Ti₃C₂ MXene was added to 10 mL of DMSO and allowed to stir for 24 h. The dispersion was then centrifuged at 3600 rpm for 10 min. The resulting solution was added to 150 mL of DI water while bubbling with argon gas, followed by sonication for 3 h. This was then centrifuged again at 3500 rpm for 1 h ²⁹. Subsequently, 20 mg of Ti₃C₂ MXene powder was completely re-dispersed in 20 mL DI water and ultrasonicated for another 30 min. The pH of the solution was adjusted at 9.0 with ammonia, and then the mixture was put into a 100 mL Teflon-sealed autoclave. The autoclave was heated at 100 °C for 6 h³⁰. Scheme S1 illustrates the process of preparation.

Synthesis of PANI@Ti₃C₂ MXene quantum dots and PANI@Ti₃C₂ MXene quantum dots -Au NPs

PANI@Ti₃C₂ MXene Quantum dots were synthesized by ultrasonically mixing 1.0 mg mL^{− 1} Ti₃C₂ MXene Quantum dots with 2.0 mg PANI. The resulting mixture was stirred at ambient temperature for 12 h. To prepare PANI@Ti₃C₂ MXene Quantum dots-Au, 500 µL of 1.0 mg mL^{− 1} PANI@Ti₃C₂ MXene Quantum Dots was added to 5 mL of freshly prepared gold nanoparticle (Au-NPs ) dispersion and mixed thoroughly. The final PANI@Ti₃C₂ MXene Quantum dots-Au nanocomposite was obtained by freeze-drying the mixture²⁶. Gold nanoparticles (Au-NPs) were synthesized via a one-step aqueous-phase reduction method using soluble starch as both the reducing and stabilizing agent, following a previously reported procedure. In a typical synthesis, 200 µL of an aqueous solution of hydrogen tetrachloroaurate (III) trihydrate (HAuCl₄·3H₂O, 0.05 mol L^{− 1}) was added to 50 mL of an aqueous solution containing 0.2 wt% soluble starch under vigorous magnetic stirring. The pH of the mixture was adjusted to pH 11.0 ± 0.2 using 0.05 mol L^{− 1} sodium hydroxide (NaOH) solution. The reaction mixture was stirred at ambient temperature for 1 h, during which a light pink hue appeared, indicating the nucleation of Au-NPs. The solution was then maintained at 70 °C for 6 h. The progression to a deep burgundy color confirmed the completion of the reduction process and the successful formation of stable colloidal gold nanoparticles³¹.

Preparation of GCE modification with PANI@Ti₃C₂ MXene quantum dots-Au composite

The sensor fabrication used a glassy carbon electrode (GCE) with a 3 mm diameter as its working electrode. The GCE surface received mechanical polishing with 0.05 μm Al₂O₃ slurry before undergoing five-minute ultrasonic cleaning sessions with ethanol and ultrapure water to remove surface contaminants. The PANI@Ti₃C₂ MXene Quantum Dots-Au nanocomposite required ultrapure water to create a 1.0 mg mL^− 1 (w/v%) stock suspension. The dispersion process involved ultrasonic treatment for thirty minutes at room temperature to achieve uniform distribution while stopping nanoparticle aggregation. The electrochemical system required ultrapure water as the dispersing solvent because it provides system compatibility and contains no interfering ions. The 1.0 mg mL^− 1 nanocomposite suspension received a 3.0 µL drop onto the cleaned GCE surface which dried naturally at room temperature for 20 min under dust-free conditions.

Immunosensor fabrication

The prostate-specific antigen (PSA) antibody (Ab₁) was covalently attached to the PANI@Ti₃C₂ MXene Quantum dots-Au/GCE via the interaction between the gold atoms in the composite and the amino (–NH₂) groups of the antibody. After preparing the PANI@Ti₃C₂ MXene Quantum Dots-Au/GCE, the electrode was immersed in an Ab1 solution (80 µg mL^− 1 in 0.01 mol L^− 1 PBS, pH 7.0) and incubated for 40 min at 37 °C. Following incubation, unbound Ab₁ was thoroughly rinsed off using PBS. To block nonspecific binding sites, the electrode was further incubated with 1% (w/w) bovine serum albumin (BSA) at 37 °C for 40 min. After rinsing with PBS to remove residual BSA, the resulting immunosensor was finalized and designated as BSA/Ab₁/PANI@Ti₃C₂ MXene Quantum dots-Au/GCE. Subsequently, varying concentrations of PSA were applied to the BSA/Ab₁/PANI@Ti₃C₂ MXene Quantum dots-Au/GCE and incubated for 40 min at 37 °C. To eliminate unbound PSA proteins, the immunosensor (PSA/BSA/Ab1/PANI@Ti₃C₂ MXene Quantum dots-Au/GCE) was washed with 0.1 mol L^− 1 PBS (pH 7.0).

Preparation of Ab₂ bioconjugates based on V₂C quantum dots@Au-NPs

V₂C MXene was synthesized by etching the V₂AlC phase. In a standard procedure, 1 g of V₂AlC powder was gradually submerged in 10 mL of HF (50 wt%) solution in a Teflon bottle and stirred vigorously at room temperature for 72 h to remove the Al layer. The resultant mixture was washed with deionized water and centrifuged at 6000 rpm for 5 min. The process was repeated several times until the supernatant reached a pH of approximately 7.0. Vacuum filtration and vacuum drying at 60 °C for 8 h were employed to yield the Layered V₂C MXene product³². Next, 0.5 g of V₂C MXene was added to 10 mL of DMSO and stirred for 24 h. Suspension was centrifuged at 3600 rpm for 10 min, and dispersion was added into 150 mL of deionized water. Argon gas was bubbled into suspension and then sonicated for 3 h. Further centrifugation at 3500 rpm for 1 h yielded the final product²⁹. The V₂C MXene Quantum dots were subsequently synthesized using the pre-prepared V₂C MXene as precursors in a hydrothermal treatment at 100 °C in aqueous ammonia solution³⁰.

The V₂C MXene Quantum Dots@Au-NPs composite was synthesized based on previously reported procedures with minor modifications. Briefly, 6 mg of V₂C MXene Quantum dots was ultrasonically dissolved in 3 mL of ultrapure water for 30 min to give a uniform solution. Next, 500 µL of HAuCl₄·3H₂O aqueous solution (30 µM) was gradually added while gently stirring. After a 10-minute reaction, the resulting V₂C MXene Quantum dots@Au-NPs suspension was centrifuged at 8000 rpm for 5 min, and the precipitate was collected³³. A total of 50 mg of V₂C MXene Quantum dots@Au-NPs was resuspended in 50 mL of ultrapure water. After 60 min, the suspension underwent several centrifugation cycles with ultrapure water. For the conjugation, 20.0 µL of Ab₂ (30.0 µg mL^{− 1}) was attached to the composite through the interaction between gold atoms and the -NH₂ groups of Ab₂. The resulting mixture was dispersed in 0.1 mol L^{− 1} PBS (pH 7.0) and stored at 4 °C (Scheme S2). A sandwich-type immunoassay was developed for the sensitive electrochemical detection of PSA Following the preparation of the PSA /BSA/Ab₁/PANI@Ti₃C₂ MXene Quantum dots-Au/GCE, 15.0 µL of the Ab₂ bioconjugate solution was applied to the electrode surface for 50 min to facilitate the immune reaction. The immunosensor (V₂C MXene Quantum dots@Au/Ab₂/PSA /BSA/Ab₁/PANI@Ti₃C₂ MXene Quantum dots-Au/GCE) was washed with PBS (0.1 mol L^{− 1}, pH 7.0) to remove unbound Ab₂ bioconjugates.

Finally, for the quantitative electrochemical detection of PSA, 20 µL of 30.0 mM H₂O₂ was added and allowed to react for 20 min. The treated electrodes were then analyzed using DPV measurements conducted in the potential range of -0.5 to 0.5 V.

Identification of MXene quantum dots

FT-IR analysis of the Titanium MXene Quantum dots is shown in (Fig. 1). As indicated in the figure, the peaks at 3494 and 3002 cm^{− 1} originated from the stretching modes of Hydroxyl and amine functional groups (O-H and N-H), respectively³⁴. The peaks at 578, 114, and 1731 cm^{− 1} correspond to the stretching vibrations of Ti-O, C-F, and C = O bonds, while the peak at 1319 cm^{− 1} corresponds to the bending vibrations of O–H^35,36,37,38.

FTIR spectra of the MXene QDs sheets.

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The morphology and microstructure of the Ti₃AlC₂ MAX phase, Ti₃C₂ MXene, Ti₃C₂ MXene (treated with DMSO), and Ti₃C₂ MXene Quantum dots were examined using Field Emission Scanning Electron Microscopy (FE-SEM). The Ti₃AlC₂ MAX phase exhibits a dense structure.

The multilayer configuration confirms the morphology of Ti₃C₂ MXenes, which were transformed into single or multilayer MXenes via ultrasonication in the presence of DMSO solvent, as well as into MXene quantum dots (Fig. 2a-d).

FE-SEM images (a,b,c,d) of the original bulk material Ti₃AlC₂, the as-obtained layered Ti₃C₂ MXene, Ti₃C₂ MXene (DMSO), and Ti₃C₂ MXene QDs; FE-SEM-EDX elemental mapping images (e, f) of the pristine Ti₃C₂ MXene QDs sheets.

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To further investigate Ti₃C₂ MXene Quantum dots, Energy-dispersive X-ray spectroscopy (EDX) analysis was used to identify their elemental compositions. EDX analysis validated the presence of carbon (C) and titanium (Ti) elements, which are characteristic of MXene Quantum dots³⁹. The EDX findings also confirmed the successful removal of the Al element, with only Ti and C elements remaining in the MXene Quantum dots nanostructure (Fig. 2e, f).

FE-SEM images of (a) the PANI@Ti₃C₂ MXene Quantum dots-Au and (b) the PANI@Ti₃C₂ MXene Quantum dots.

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Additionally, the PANI@Ti₃C₂ MXene QDs-Au and PANI@Ti₃C₂ MXene Quantum dots (Fig. 3a, b), as well as the V₂AlC MAX phase, V₂C MXene, V₂C MXene (treated with DMSO), and V₂C MXene Quantum dots composites were examined using FE-SEM. The MAX phase exhibited a dense structure, while the MXene phases displayed a layered structure (Fig. 4a-c).

FE-SEM images of (a) the V₂AlC MAX phase, (b) the V₂C MXene, and (c) the V₂C MXene (DMSO).

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Figure 5a, b shows the layered structure of MXene quantum dots, as well as the presence and uniform distribution of gold nanoparticles. To further investigate V₂C MXene quantum dots, EDX analysis was used to identify its elemental compositions. The presence of carbon (C) and vanadium (V) elements was confirmed (Fig. 5c).

FE-SEM images (a,b) of the V₂C MXene Quantum dots and V₂C MXene Quantum dots-Au NPs; FE-SEM-EDX elemental mapping images (c) of the pristine V₂C MXene QDs sheets.

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TEM images of MXene quantum dots in (Fig. 6a, b) show that they are uniformly dispersed without significant aggregation. The average size of the Ti₃C₂ MXene Quantum dots was estimated to be 3.29 ± 0.06 nm, while the size of the V₂C MXene quantum dots is about 2.92 nm, as shown in the histogram of the MXenes, confirming that the MXenes are indeed quantum dots (Fig. 6c, d). X-ray diffraction (XRD) analysis was used to determine the nature of Ti₃C₂ MXene Quantum dots by providing information about their crystallographic structure, lattice parameters, and phase composition.

TEM images (a,b) and histograms (c,d) of Titanium and Vanadium quantum dots of MXenes, respectively.

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The XRD patterns of the Ti₃AlC₂ MAX phase and MXene QDs.

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The XRD analysis also illuminated the atomic arrangement of the Ti₃C₂ MXene Quantum dots. (Fig. 7) illustrates that the Ti₃C₂ MXene Quantum dots exhibit a layered structure, which is similar to that of other layered MXenes. Following the etching of the MAX phase, the prominent (1 0 4) peak of Ti₃AlC₂ at 39.0° disappears in the Ti₃C₂ MXene⁴⁰. Additionally, the broadening of diffraction peaks in Ti₃C₂ MXene Quantum dots compared to their bulk counterparts indicates a reduction in crystallite size, a hallmark of quantum confinement effects⁴¹. The XRD patterns of Ti₃C₂ MXene Quantum dots reveal an increase in interlayer distance owing to the removal of intercalated species and the incorporation of functional groups such as hydroxyl (-OH) and fluoride (F⁻). The shift of the (0 0 4) peak towards higher angular values signifies accordion-like structural changes brought about by hydrothermal treatment. These changes enhance the electrochemical properties of Ti₃C₂ MXene Quantum dots, which make them extremely suitable for biosensing purposes⁴².

In (Fig. 8a, b) depict the CV and electrochemical impedance spectroscopy (EIS) curves obtained during the stepwise fabrication of the immunosensor in a 5 mM [Fe(CN)₆]^3−/4− solution with 0.1 mol L^− 1 KCl. These electrochemical techniques provide key insights into the electrode modification process, confirming successful sensor construction and assessing electron transfer dynamics.

To improve the biosensor’s electrochemical performance, polyaniline (PANI) was combined with MXene quantum dots to create a conductive and functional composite interface. The main interaction mechanisms include hydrogen bonding between the -NH₂ and -NH groups of PANI and the surface terminations (-OH, O²⁻, F^ˉ) of MXenes. There is also π-π stacking between the conjugated PANI backbone and the structure less MXene basal planes, along with electrostatic stabilization from the positively charged PANI matrix interacting with the negatively charged MXene surfaces. These combined forces lead to better dispersion, increased electron transfer, and higher redox activity of the nanocomposite. The PANI@MXene configuration increases the interface area of this biosensor. CV analysis is a critical method for the description of redox activity of modified electrodes. Bare GCE has very low peak currents, indicating limited electron transfer kinetics. Peak currents are dramatically increased following modification with the PANI@Ti₃C₂ MXene quantum dots-Au nanocomposite, indicating the enhanced efficiency of electron transfer through the conductivity of MXene quantum dots and gold nanoparticles (Fig. 8a). Ab₁ (primary antibody) binding resulted in a reduction of the current response. This is because of the insulating nature of biomolecules, which prevents the flow of electrons on the electrode surface. Further reductions in current intensity are observed with bovine serum albumin (BSA) immobilization and PSA antigen, again demonstrating these biomolecules to be charge-blocking agents⁴³.

Research shows that polyaniline (PANI) plays an important role in the conductivity of the electrode and also provides active sites for the electrochemical process via its redox-active backbone. Ti₃C₂TX, on the other hand, has high metallic conductivity, high surface area and a large number of surface terminations (–OH, O²⁻, F^ˉ) which can easily make the electrons move quickly and also immobilize the biomolecules. The MXenes, as opposed to PANI, have a different structure of layers which allow for ion intercalation and fast charge transport, thus, reducing the interfacial resistance. Moreover, PANI can help with the film-forming ability and the mechanical flexibility of the electrode, while MXenes are responsible for the charge transfer kinetics, the electrochemical stability and the catalytic activity which makes them the main source of biosensor sensitivity improvement^44,45,46. EIS is a highly sensitive method for evaluating the interfacial charge transfer resistance (Rct) of electrochemical systems. In the Nyquist plots presented in (Fig. 8b), the semicircular diameter corresponds to the Rct value, reflecting changes in surface conductivity during immunosensor construction Initially, the bare GCE, which has a large Rct, corresponds to a simple surface with low conductivity. Upon deposition of the PANI@Ti₃C₂ MXene Quantum dots-Au NPs composite, there is a significant decrease in impedance, as per the conductive nature of MXenes and gold nanoparticles, which favor electron transfer. The sequential functionalization with Ab1, BSA and PSA antigen, the Nyquist semicircle grows progressively, as a sign of an increase in Rct. This trend again verifies the insulating nature of biomolecular layers and confirms step-by-step, successful assembly of the immunosensor^47,48.

CV (a) EIS (b) curves obtained from different electrodes in the fabrication of the immunosensor in [Fe (CN)₆] ^3−/4− solution. and Effect of pH on the immunosensor response (c, d).

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Optimization of immunosensor conditions

Several factors, including the pH of the buffer, concentrations of H₂O₂ and Ab₂, and incubation times for Ab₁ and Ab₂, significantly influence the immunosensor’s electrochemical performance. These parameters were optimized as follows:

The pH of the phosphate buffer is highly important in the electrochemical behavior of the immunosensor as it directly impacts the activity and stability of the immobilized proteins. The establishment of the pH conditions which are the best ones for the maximum antigen-antibody interaction is crucial because that way the immunosensor becomes more sensitive and reliable. Electrochemical Response to pH alterations: The CV and DPV measurements were conducted using 0.1 mol L^{− 1} phosphate buffer solutions of differing pH between the range 3.0–10.0 (Fig. 8c, d). The data obtained, it has been observed the current response rises from pH 3.0–7.0, showing that there is heightened protein activity with improved electron transfer kinetics. This behavior arises from the fact that the intermediate ionized state of the antibody-antigen complex enables the most effective way toward the interface for the electrochemical charge transfer. Moreover, under very high alkaline conditions (pH > 8.0), the bio-components lost the originality of the structural form and the response of electron transfer diminished. Thus, for the best immunosensor performance, 7.0 pH was selected to be the optimum condition for the detection procedure^49,50.

The incubation time for Ab₁ and Ab₂ was examined to identify the optimal duration for the immunoreaction. For Ab₁, incubation times ranging from 10 to 70 min were tested (Figure S1a). The inhibition ratio of the current response increased with longer incubation times, peaking at 40 min. Beyond this point, the inhibition ratio declined, likely due to the detrimental effects of prolonged exposure or temperature on biomolecular function. Consequently, an incubation time of 40 min was deemed optimal for Ab₁.

Similarly, the response of the immunosensor to Ab₂ conjugated with V₂C MXene quantum dots @Au was analyzed at varying incubation times (Figure S1b). An incubation time of 40 min was also found to be ideal for the Ab₂ bioconjugate interaction.

The concentration of Ab₂ conjugated with V₂C MXene quantum dots@Au nanoparticles was optimized to maximize signal amplification. As shown in (Figure S1c), the peak current increased with Ab₂ concentrations up to 30 µg mL^− 1, after which it plateaued or slightly diminished. Thus, 30 µg mL^− 1 was selected as the optimal Ab₂ concentration.

Hydrogen peroxide (H₂O₂) is a important compound as the redox probe in electrochemical immunosensors, which is involved in the catalytic reaction and the signal amplification. The adjustment of its concentration to time is a must for great sensitivity and the efficiency of detecting PSA. The determination of the correlation between the concentration of H₂O₂ and immunosensor’s performance was done using the technique of DPV (Figure S1d). The current response increased with H₂O₂ concentration up to 30 mM, which means a better catalytic activity but most importantly it shows a faster electron transfer between the analyte and the electrode. At concentrations of 30 mM and above, the surplus of the H₂O₂ led to a low effect of the catalytic reaction so the current also fell down. This reduction might be understood as really high H₂O₂ concentrations that caused oxidative stress to the biomolecular components; that is, oxidative stress jeopardized both enzymes and the electrode surface. Consequently, 30 mM H₂O₂ was determined to be the optimal concentration for achieving maximum immunosensor performance⁵¹.

Validation

Blood specimens from healthy donors were collected from Shahid Jalil Hospital in Yasouj, Iran, and stored frozen until used. Detection of PSA by the immunosensor was done by diluting the samples at a 1:1 ratio with distilled water. To simulate conditions in cancer patient with elevated PSA levels, varying concentrations of standard PSA solutions were spiked into the serum samples. Using the standard addition method, recoveries of 100.00% to 100.50% were obtained, exhibiting very high accuracy.

The electrochemical abilities of the immunosensor constructed were evaluated by DPV in the presence of different PSA concentrations. As depicted in Fig. 9a, a clear trend was observed: the oxidation peak current decreased proportionally with increasing PSA concentration, confirming the high sensitivity of the immunosensor.

(a) DPV curves of the immunosensor in the presence of different concentration (2 fg mL^− 1 to 2 pg mL^− 1) PSA, (b) the calibration curve for determination of PSA, (c) the currents obtained on the developed immunosensor, and (d) inter-day measurements for the stability of the prepared immunosensor.

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The reduction mentioned was due to the biomolecular layers being insulative in nature, which were formed during the sandwich-type immunoassay, and thus progressively blocked the electron transfer at the electrode interface. Along with the current shifts, there was a prominent anodic potential shift at the peak. The change in peak potential originates from the changed interface dynamics due to the repeated immunoreactions. By the time the PSA antigen is caught by Ab₁ and connected with Ab₂@V₂C MXene QDs@Au-NPs nanoconjugate, the electrode surface that is inactive from the point of view of the electrochemical reactions gets covered with biomolecular layers more and more. The sequential layering leads to charge redistribution, increased steric hindrance, and changed electron transfer routes. All these raise the overpotential that is needed for the oxidation of the electroactive species (i.e., H₂O₂), resulting in the observed Ep shift. This case shows the signal decrease not only as a result of the lower conductivity but also due to the changing electrochemical impedance at the interface. These shifts in peak potential have been observed in biosensors in which restricted electron mobility as a result of high-density surface functionalization and interfacial electrostatics alter the redox potential of the system^47,48,51. The electrochemical sensitivity of the device are thus maintained through this alteration, also provides a further qualitative indication of successful antigen capture and immunocomplex formation. In Fig. 9b, the calibration plot with a very low detection limit of 0.61 fg mL^{− 1} (S/N = 3) is demonstrated, showing the linearity between peak current and PSA concentration in the range 2 fg mL^{− 1} to 2 pg mL^{− 1}.

These metrics strongly affirm the sensor’s suitability for ultra-trace biomarker detection. In future iterations, both current amplitude and peak potential shift may be co-utilized to enhance dual-signal validation protocols in biosensing applications. Comparisons with other electrochemical PSA immunoassays (Table 1) indicate that the developed sensor performs comparably or better in certain instances.

Reproducibility was assessed using intra-assay and inter-assay variations. For intra-assay precision, PSA analysis was performed on three immunosensors, with six replicates each (Table 2). The relative standard deviation (RSD) values reflect a excellent repeatability of the immunosensor.

The immunosensor’s potential in the PSA detection using human serum was confirmed with the standard addition method. The samples were diluted to 50-fold and spiked with differing PSA concentrations. The results ranged from 100.125% to 100.286% at + 100 mV (Table 3) confirming the method’s accuracy, precision, and specificity for real-world applications.

The principle of selectivity was examined by challenging different immunosensor samples with five biomarkers mixtures, containing IgG, streptavidin (STR), carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), and a mixture of them all. The solutions were as follows 0.50 pg mL^− 1 PSA, 1.50 pg mL^− 1 CEA, IgG, STR, and AFP in 0.1 mol L^− 1 phosphate-buffered saline (pH 7.0). The results were unambiguously in favor of the immunosensor’s selectivity for PSA detection, as non-PSA biomarkers exhibited no change in the measurements (Fig. 9c).

The assessment of stability was done by keeping the immunosensors at 4 °C for a period of 30 days. Besides the current response they got by using the 0.5 pg mL^− 1 PSA concentration, the responses which were obtained after the 30-day storage visualized 96.34% of its initial value, thus assuring a satisfactory stability of the immunosensor (Fig. 9d). To check repeatability, 30 consecutive voltammograms were recorded in the presence of 20.0 mM H₂O₂. The responses at + 100 mV were highly consistent, with an RSD of 0.237, which confirmed the reliability of the immunosensor.

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Authors and Affiliations

1. Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran

   Mohammad Safarpoor & Rassoul Dinarvand

2. Medicinal Plants Research Center, Yasuj University of Medical Sciences, Yasuj, Iran

   Arash Asfaram

3. Chemistry Department, Yasouj University, Yasouj, 75918-74831, Iran

   Mehrorang Ghaedi

Contributions

M. S.: Methodology, Investigation, Software; Formal analysis; Writing – Original Draft, Writing – review & editing A. A.: Methodology, Validation, Formal analysis, Investigation, Writing – original draft, Writing – review & editing. M. G.: Supervision, Methodology; Writing – review & editing, Methodology; Validation, Investigation., R. D.: Supervision, Conceptualization, Methodology; Writing – original draft, Writing – review & editing, Validation, Project administration; Resources; Funding acquisition.,

Corresponding authors

Correspondence to Mehrorang Ghaedi or Rassoul Dinarvand.

Competing interests

The authors declare no competing interests.

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