An Integrated Electrochemical Platform Based on Ti 3 C 2 -Ag NPs and 3D- Printed Microfluidics for Simultaneous Detection of PSA and PSMA

An Integrated Electrochemical Platform Based on Ti 3 C 2 -Ag NPs and 3D- Printed Microfluidics for Simultaneous Detection of PSA and PSMA | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article An Integrated Electrochemical Platform Based on Ti 3 C 2 -Ag NPs and 3D- Printed Microfluidics for Simultaneous Detection of PSA and PSMA Song-Song Yang, Lu Han, Wu-Lin Xin, He-Qing Cai, Kou Zhang, Xin-Yu Xue, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7470190/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Feb, 2026 Read the published version in Microchimica Acta → Version 1 posted 16 You are reading this latest preprint version Abstract The multiplexed detection of tumor biomarkers represents a transformative strategy to improve diagnostic accuracy in oncology. A significant to the clinical translation of multiplexed label-free electrochemical immunosensors (EIs) is the issue of analytical reliability, which is frequently compromised by signal crosstalk and insufficient sensitivity. In this work, we designed a 3D-printed, reconfigurable microwell array through a combination of screen-printing, stereolithography (SLA) 3D printing, and microfluidic technologies for interference-free multiplexed detection. To achieve high sensitivity, the electrode surfaces were modified with a novel Ti 3 C 2 -Ag NPs nanocomposite that significantly enhances charge transfer kinetics by preventing Ti 3 C 2 nanosheet aggregation through modulation of Ag NPs interlayer spacing. This integrated platform was validated through the simultaneous quantification of two critical prostate cancer biomarkers, prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA). The immunosensor demonstrated both the complete elimination of signal cross-talk and excellent analytical performance, including a wide linear range (0.1–1,000 ng·mL⁻¹), low sensitivities (0.0036 µA·mL·ng − 1 for PSA and 0.0024 µA·mL·ng − 1 for PSMA), and low limits of detection (0.045 ng·mL⁻¹ for PSA and 0.041 ng·mL⁻¹ for PSMA). Furthermore, this device exhibited exceptional repeatability, stability, and specificity. Clinical validation using human serum samples exhibited strong concordance with clinical reference methods, enabling precise discrimination between prostate cancer patients and healthy controls. Consequently, the proposed dual-channel label-free EI, based on Ti 3 C 2 -Ag NPs nanocomposites, holds substantial promise for clinical diagnostic applications, with potential for expansion to the ultrasensitive detection of other disease-related biomarkers. Dual-channel electrochemical immunosensors Multiplexed detection Ti3C2-Ag NPs nanocomposites Screen-printed electrodes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction Label-free electrochemical immunosensors (EIs) have emerged as a transformative technology for cancer biomarker detection, offering unparalleled advantages in sensitivity, specificity, rapid response, cost-effectiveness, and user-friendliness [ 1 , 2 ]. Despite these merits, conventional single-channel EIs, which target individual biomarkers, suffer from high rates of false positives/negatives in clinical settings, limiting their diagnostic reliability [ 3 ]. To address this critical gap, the development of multi-channel EIs has gained momentum, promising enhanced diagnostic accuracy, reduced analysis time, and lower operational costs through simultaneous multi-analyte detection. A typical multi-channel EI system integrates a reference electrode, an auxiliary electrode, and multiple working electrodes. However, signal interference (cross-talk) between adjacent working electrodes primarily caused by diffusion of electroactive species—remains a significant technical hurdle [ 4 ]. While prior strategies, such as minimizing electrode surface area [ 5 ] and increasing inter-electrode spacing [ 6 ], mitigate cross-talk, they inadvertently compromise sensor performance by elevating the limit of detection (LOD) or reducing sensitivity. Thus, innovative approaches are urgently needed to resolve this trade-off without sacrificing analytical efficacy. Screen-printed electrodes (SPEs), serving as the central component of label-free EIs, offer significant advantages, notably their cost-effectiveness, user-friendly operation, and scalability [ 7 ]. Advanced screen-printing techniques enable precise fabrication of disposable electrodes with micron-scale resolution. Furthermore, SPEs can be tailored to meet the specific requirements of various types of EIs, allowing for the creation of diverse patterns through layering techniques. Consequently, novel EIs can be developed with confidence by leveraging the versatility of SPEs [ 8 ]. The performance of these systems is critically dependent on electrode materials, because appropriate electrode materials can offer a substantial specific surface area for immobilizing a greater number of antibodies and superior electrical conductivity to facilitate electron transfer on the electrode surface [ 9 , 10 ]. Two-dimensional (2D) layered nanomaterials, including transition metal sulfides [ 11 ], black phosphorus [ 12 ], layer double hydroxide (LDH) [ 13 ], and MXenes [ 14 ] have gained widespread use owing to their unique layered structures, large specific surface areas, ultra-thin nature, excellent physicochemical properties, and thickness-dependent band gaps [ 15 ]. Among these, MXenes offer exceptional versatility through tunable M/X element combinations and surface functionalization [ 16 ], exemplified by the metallic conductivity of Ti 3 C 2 -MXene [ 17 ]. However, their intrinsic 2D structures are prone to restacking, hindering electron transfer on the electrode surface of EIs and limiting their electrochemical performance [ 18 ]. Recent advances demonstrate that MXene nanocomposites, which composite conductive materials including noble metal nanoparticles (MNPs), carbon nanostructures, and conductive polymers via the numerous functional groups (-OH, -F, =O, etc.) on the surface, can overcome these limitations through synergistic interfacial interactions [ 19 ]. Among MNPs, gold nanoparticles (Au NPs) and silver nanoparticles (Ag NPs) are particularly versatile and widely employed in biosensing, catalysis, and medical imaging due to their nanoscale effects and excellent electrical conductivity [ 20 ]. Ag NPs, in particular, offer exceptional cost-effectiveness, making them the preferred choice for reducing the production costs of MXene-based nanocomposites. Here, we address these challenges through a dual-pronged approach. Firstly, we leverage SPEs to fabricate a three-electrode system with dual working electrodes modified by Ti 3 C 2 -Ag NPs nanocomposites. This design amplifies electrochemical signals while maximizing antibody immobilization density. Secondly, we propose an innovative approach that combines microfluidic [ 21 ] and stereolithography (SLA) 3D printing technologies to create polydimethylsiloxane (PDMS) microfluidic channels with spatially isolated reaction chambers to eliminate cross-talk between electrodes. This architecture ensures precise localization of prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) antibodies on distinct working electrodes, enabling simultaneous and interference-free detection. The LOD values achieved for the developed dual-channel label-free EIs were 0.045 ng·mL-1 for PSA and 0.041 ng·mL-1 for PSMA, displaying their high sensitivity, specificity, and low detection limits. Finally, the sensors were used for clinical detection and quantification of PSA and PSMA in serum samples, which exhibit consistent results compared with a specialized instrument, demonstrating this dual-channel electrochemical sensor has been validated as a reliable tool for early prostate cancer (PCa) diagnosis. 2 Materials and methods 2.1 Materials PSA, PSMA, albumin, and lithium fluoride (LiF) were acquired from Sigma-Aldrich. PSA antibody and PSMA antibody were procured from Cell Signaling Technology. Aluminum titanium carbide (Ti 3 AlC 2 ), bovine serum albumin (BSA), glucose, and phosphate-buffered saline (PBS) were purchased from Shanghai Macklin Biochemical Co., Ltd. Silver paste, carbon paste, and insulating ink were obtained from Jujo Chemical Co., Ltd. Polyethylene terephthalate (PET) film (100 µm) and PDMS were sourced from Beijing Chemical Plant. All other reagents were of analytical grade and employed without further purification. Clinical serum samples were obtained from Peking University Shougang Hospital (Beijing, China). Ethics Committee of Peking University Shougang Hospital approved the use of this material for research purposes, and informed consent was provided from all patients (approval number: 23587-0-01). 2.2 Synthesis of Ti 3 C 2 nanosheets As shown in sheme.1A, layered Ti 3 C 2 -MXene was exfoliated via minimally intensive layer delamination [ 22 ]. Briefly, 1.6 g LiF was dissolved in 20 mL HCl (15 mL 37% HCl + 5 mL H 2 O), followed by gradually add 1 g of Ti 3 AlC 2 and stir the reaction (38 ° C, 48 h). The product was centrifuged (3,500–8000 rpm, 10 min) and washed with ultra-pure water multiple times until the pH of the supernatant reached approximately 6. The bottle-green supernatant containing Ti 3 C 2 nanosheets was collected and subsequently freeze-dried for further testing. 2.3 Preparation of Ti 3 C 2 -Ag NPs nanocomposites Ti 3 C 2 -Ag NPs nanocomposites were synthesized using a self-reduction method (Sheme.1A). Ti 3 C 2 suspension (5 mL) was mixed with 5 mL H 2 O, followed by dropwise addition of 200 µL 0.1 M AgNO 3 under stirring at 25 ºC (1000 rpm, 1 h). Centrifuge (8,000 rpm, 10 min) and collect the supernatant to obtain the Ti 3 C 2 -Ag NPs nanocomposites for further characterization and application. 2.4 Characterization of Ti 3 C 2 and Ti 3 C 2 -Ag NPs nanocomposites The structures of the as-prepared Ti 3 C 2 nanosheets and Ti 3 C 2 -Ag NPs nanocomposites were characterized using Ultraviolet-visible (UV-vis, UV-2501PC, SHIMADZU, Japan), Fourier transform infrared (FTIR, IS-5, Thermo Scientific, USA), X-ray diffractometer (XRD, Bruker D8, Bruker AXS, Germany), and Raman spectroscopy (Xplora-BX51, HORIBA, France). The microstructural and morphological features of the as-prepared Ti 3 C 2 nanosheets and Ti 3 C 2 -Ag NPs nanocomposites were examined using scanning electron microscopy (SEM, SU8020, HITACHI, Japan) and transmission electron microscopy (TEM, F20, FEI, Netherlands).The conductivity was measured using a four-point probe system (RTS-9, Guangzhou, China) and surface profiler (Dektak 150, Veeco, USA) and the specific operation is recorded in the supporting information. 2.5 Construction of the dual-channel label-free EIs Scheme.1 Preparation route of Ti 3 C 2 -Ag NPs nanocomposites and the dual-channel label-free EIs (A); Application of PCa diagnosis based on the dual-channel label-free EIs (B) 2.6 Electrochemical properties The electrochemical performance of all samples was evaluated using an electrochemical workstation (AutoLab, Metrohm, Switzerland). Cyclic voltammetry (CV) and square wave voltammetry (SWV) were performed in 0.01 M PBS (pH 7.4) containing 5 mM K 3 [Fe(CN) 6 ], 5 mM K 4 [Fe(CN) 6 ], and 0.1 M KCl. CV tests were conducted in the potential range of -0.2 V to 0.6 V at a scanning rate of 100 mV·s − 1 , while SWV tests were performed in the potential range of -100 mV to 600 mV at an amplitude of 20 mV. Incubate (25° C, 3 h) 3 µ L of PSA and PSMA diluted solutions of different concentrations (0.1–1000 ng·mL⁻ 1 ) on the corresponding electrodes of dual-channel label-free EIs, wash multiple times, and analyze the immunosensor using CV and electrochemical impedance spectroscopy (EIS) to evaluate sensitivity, linear dynamic range, repeatability, stability, and specificity. The frequency range measured by EIS is 0.1 Hz to 1 MHz, with an amplitude of 120 mV. 2.7 Detection of PSA and PSMA in clinical samples To assess clinical applicability, serum levels of PSA and PSMA were quantified in four anonymized human samples using the developed dual-channel label-free EIs. Briefly, serum samples from 1 healthy donor and 3 prostate cancer patients was diluted 1:10 in PBS. Samples (3 µL) were incubated on antigen-specific electrodes (37°C, 30 min), washed, and analyzed via CV. The specific operation is recorded in the supporting information. 3 Results and Discussion 3.1 Characterization of Ti 3 C 2 nanosheets To confirm the successful exfoliation of the "Al" layer from Ti 3 AlC 2 , UV-vis, FTIR, XRD, and Raman spectroscopy were employed to characterize the Ti 3 C 2 nanosheets, the results of which were shown in Fig. S1 . The SEM images in Fig. 1 A and B reveal the presence of a distinct layered structure in Ti 3 AlC 2 on silicon wafers, with the layers tightly adhering to each other. However, after undergoing MILD treatment, Ti 3 AlC 2 transforms into a highly thin, smooth, and flaky sheet structure, as depicted in Fig. 1 C and D. Additionally, the folding and stacking of these ultra-thin Ti 3 C 2 -MXene sheets are evident, further confirming the successful production of mono- or few-layered Ti 3 C 2 . The significantly larger specific surface area of the ultra-thin Ti 3 C 2 , compared to Ti 3 AlC 2 , facilitates the binding of Ag NPs, thereby enhancing the electrochemical performance of Ti 3 C 2 -MXene. 3.2 Characterization of Ti 3 C 2 -Ag NPs nanocomposites To determine whether Ag NPs were effectively anchored onto Ti 3 C 2 nanosheets, UV-vis and XRD and TEM analyses were conducted. The surface plasmon resonance (SPR) characteristic peak of Ag NPs at 425 nm (Fig. 2 A) and the (111) crystal face peak of Ag NPs at 39° (Fig. 2 B) were both observed in Ti 3 C 2 -Ag NPs nanocomposites, indicating successful integration of Ag NPs with Ti 3 C 2 . A typical TEM image of mono- or few-layered Ti 3 C 2 -MXene with gauze-like sheets is shown in Fig. 2 C. After sufficient reaction with AgNO 3 , a large number of uniformly distributed Ag NPs with small particle size were observed on the surface of Ti 3 C 2 sheets (Fig. 2 D), providing further evidence of successful preparation of Ti 3 C 2 -Ag NPs nanocomposites. This can be attributed to the residual = O, -OH, and -F moieties on the surface of Ti 3 C 2 nanosheets, which provided a sufficient number of chemically active sites for deposition of Ag NPs. 3.3 Electrochemical properties of Ti 3 C 2 -Ag NPs nanocomposites To investigate the impact of "Al" exfoliation on the conductivity of Ti 3 C 2 , we measured the electrical conductivity of Ti 3 AlC 2 and Ti 3 C 2 using a four-point probe resistivity measurement system. As shown in Fig. 3 A, the conductivity of Ti 3 C 2 was found to be 3989 S·cm − 1 , which is 15 times higher than that of Ti 3 AlC 2 (257 S·cm − 1 ) and consistent with previously reported literature [ 23 ]. To determine whether the integration of Ag NPs with Ti 3 C 2 could enhance the electrical conductivity of Ti 3 C 2 , we immobilized as-synthesized Ti 3 C 2 nanosheets and Ti 3 C 2 -Ag NPs nanocomposites onto SPEs and measured their CV and SWV curves. As shown in Fig. 3 B, Ti 3 C 2 /SPE exhibited a larger redox peak current and a smaller potential difference than bare SPE due to the accelerated electron transfer effect by Ti 3 C 2 nanosheets. The redox peak current of Ti 3 C 2 -Ag NPs/SPE continued to increase significantly, and the potential difference of Ti 3 C 2 -Ag NPs/SPE further decreased, indicating that the incorporation of Ag NPs onto Ti 3 C 2 nanosheets could further enhance the electron transfer on Ti 3 C 2 nanosheets. Furthermore, SWV curves (Fig. 3 C) demonstrated a significantly higher peak current for Ti 3 C 2 -Ag NPs/SPE compared to bare SPE and Ti 3 C 2 /SPE. The peak current of Ti 3 C 2 -Ag NPs/SPE was 2.38 times higher than that of Ti 3 C 2 /SPE, suggesting that Ti 3 C 2 -Ag NPs can effectively enhance the electron transfer rate on the surface of SPE, which was consistent with the CV results (Fig. 3 B). These results indicate that the electrical conductivity of Ti 3 C 2 can be remarkably enhanced by the insertion of Ag NPs, making it a promising electrode material for the construction of label-free EIs. To determine the optimal amount of Ti 3 C 2 -Ag NPs for electrode modification, various concentrations (50, 100, 150, 200, 300, 400, and 500 µg·mL-1) of Ti 3 C 2 -Ag NPs solution (3 µL) were deposited onto SPE(a) and SPE(b) electrodes, and their corresponding CV curves were recorded. As shown in Fig. 3 D and E, the peak current exhibited a continuous increase with increasing concentrations of Ti 3 C 2 -Ag NPs from 0 to 150 µg·mL-1, accompanied by a decrease in the peak potential difference. These observations indicate an enhanced electron transfer rate. However, beyond the concentration of 150 µg·mL-1, the peak current gradually decreased, and the potential difference increased. Consequently, the optimal modification amount of Ti 3 C 2 -Ag NPs onto SPEs was determined to be 150 µg·mL-1. 3.4 Electrochemical analysis of dual-channel label-free EIs A dual-channel label-free EI for the detection of PSA and PSMA was developed using Ti 3 C 2 -Ag NPs nanocomposites as highly efficient substrate materials. The changes in electrochemical behavior of the BSA/Anti-PSA/Ti 3 C 2 -Ag NPs/SPE sensor and BSA/Anti-PSMA/Ti 3 C 2 -Ag NPs/SPE sensor at each assembly step were investigated using ferricyanide as an indicator, as depicted in Fig. 4 A and D. It can be observed that the redox peak currents of the SPEs were significantly enhanced, and the redox potential difference was remarkably reduced after the modification with Ti 3 C 2 -Ag NPs nanocomposites. Subsequently, a gradual decrease in the peak current was observed after the sequential immobilization of Anti-PSA (or Anti-PSMA), BSA, and the corresponding PSA (or PSMA), indicating the successful immobilization of these molecules onto the dual-channel label-free EI and the inhibition of electron transfer on the SPEs surfaces [ 24 ]. The incorporation of Ti 3 C 2 -Ag NPs amplified the response of the fabricated label-free EIs, leading to an increase in the peak current, as Ti 3 C 2 -Ag NPs serve as efficient conductors to enhance the signal. EIS is a powerful technique for monitoring changes in the surface properties of modified electrodes. The EIS typically consists of a semicircular region corresponding to the electron transfer process at higher frequencies and a linear region corresponding to the electron diffusion process at lower frequencies. The diameter of the semicircle represents the electron-transfer resistance (R et ) [ 25 ]. In Fig. 4 B and E, the corresponding EIS spectra of the developed BSA/Anti-PSA/Ti 3 C 2 -Ag NPs/SPE sensor and BSA/Anti-PSMA/Ti 3 C 2 -Ag NPs/SPE sensor at different stages are presented. The bare SPE exhibited the highest resistance. However, upon modification with Ti 3 C 2 -Ag NPs, a significant decrease in the R et value was observed, indicating enhanced electron transfer facilitated by Ti 3 C 2 -Ag NPs [ 26 ]. Subsequently, a gradual increase in R et values was observed after sequential modification of Anti-PSA (or Anti-PSMA), BSA, and PSA (or PSMA) onto SPE(a) (or SPE(b)), which can be attributed to the successful immobilization of these protein molecules. The formation of the antibody-antigen immunocomplex on the electrode interface hindered charge transfer [ 27 ]. These results further confirm the successful fabrication of the dual-channel label-free EIs, which can be utilized for the detection of PSA and PSMA. The LOD values for the label-free EIs were determined by analyzing a series of antigens with varying concentrations [ 28 ]. The calibration plot and logarithmic calibration curve for PSA and PSMA detection under optimal experimental conditions are presented in Fig. 4 C and F. The current response exhibited a decrease with increasing concentrations of PSA and PSMA, and a linear relationship was observed between the change in current (Δi) and the logarithm of PSA and PSMA concentrations within the range of 0.1 to 1,000 ng·mL − 1 . The linear equation for the BSA/Anti-PSA/Ti 3 C 2 -Ag NPs/SPE sensor was determined as 6.25291 + 1.96397x, with a correlation coefficient of 0.99736. Similarly, the linear equation for the BSA/Anti-PSMA/Ti 3 C 2 -Ag NPs/SPE sensor was found to be 5.09592 + 2.45162x, with a correlation coefficient of 0.99673. The LOD values of the sensor were calculated using the formula X b +KS b =algC L +b, where X b represents the average peak current change value of the bare electrode, S b is the standard deviation of the peak current change value of the bare electrode, K is the signal-to-noise ratio, and C L is the LOD of the sensor [ 29 , 30 ]. Consequently, the LOD of the dual-channel label-free EIs for PSA was determined to be 0.045 ng·mL − 1 , with a linear detection range of 0.1-1,000 ng·mL − 1 and a sensitivity of 0.0036 µA·mL·ng − 1 at a signal-to-noise ratio of 3. For PSMA, the LOD of the dual-channel label-free EIs was calculated as 0.041 ng·mL − 1 , with a linear detection range of 0.1-1,000 ng·mL − 1 and a sensitivity of 0.0024 µA·mL·ng − 1 at a signal-to-noise ratio of 3. Importantly, the serum PSA concentrations of both normal individuals (4 ng·mL − 1 ) and prostate cancer patients (10 ng·mL − 1 ) fall within the linear range of the dual-channel label-free Eis [ 31 ]. Therefore, this study presents a novel method for simultaneous detection of PSA and PSMA concentrations, highlighting the potential clinical application of the developed dual-channel label-free EIs [ 1 , 32 ]. Table S1 presents a comparative analysis of proposed label-free EIs designed for the detection of PSA or PSMA. Previous studies have primarily focused on detecting either PSA or PSMA alone, limiting their applicability. In contrast, the EIs developed in this study enables the simultaneous detection of both PSA and PSMA at a lower LOD and a wide detection range. This advancement significantly enhances the accuracy of clinical analysis. While an electrochemical sensor for PSA and PSMA detection has been reported previously [ 33 ], its detection ranges is limited, and the LOD and detection ranges for PSA and PSMA are not aligned due to interference from different biomarker signals. In contrast, the label-free EIs in this work not only circumvent detection errors caused by signal crosstalk from the dual-channel working electrode, but also offer a broader detection range. More importantly, the EIs demonstrate a sensitivity of 0.0036 µA·mL·ng − 1 and 0.0024 µA·mL·ng − 1 at the picomolar level for the detection of PSA and PSMA, respectively, across a reasonably broad concentration range. This capability enables rapid cancer diagnosis and holds promise for diverse biomedical applications. 3.5 Repeatability, stability and specificity of the dual-channel label-free EIs Repeatability is a crucial factor for biosensors, and it is important to verify the reliability of our developed label-free EIs. To this end, we randomly selected five samples from four different batches and performed CV tests [ 34 ]. As shown in Fig. 5 A, the current response of the PSA sensor exhibited a relative standard deviation (RSD) of 1.83%, indicating excellent repeatability. Stability is another essential characteristic of biosensors. To evaluate the stability of the BSA/Anti-PSA/Ti 3 C 2 -Ag NPs/SPE sensor [ 35 ], we stored the sensors at 4 ℃ and examined their current responses every three days. Figure 5 B illustrates that the BSA/Anti-PSA/Ti 3 C 2 -Ag NPs/SPE sensor displayed a decrease in current response over time. After 3, 6, 9, and 12 days of storage, the current response of the sensor decreased by 6.78%, 14.18%, 14.68%, and 16.25%, respectively. After nearly two weeks of storage, the current response of the PSA sensor reached 83.75% of its original value, indicating good long-term stability of the BSA/Anti-PSA/Ti 3 C 2 -Ag NPs/SPE sensor. Furthermore, to investigate the specificity of the BSA/Anti-PSA/Ti 3 C 2 -Ag NPs/SPE sensor, we incubated the sensor with PSA in the presence of interfering agents, including Albumin, glucose, and BSA [ 36 ]. As depicted in Fig. 5 C, the average current responses of the sensors exposed to interfering agents (Albumin 5.57%, glucose 2.42%, and BSA 2.77%) were within 4% of the immunosensor without interference, demonstrating the outstanding specificity of the BSA/Anti-PSA/Ti 3 C 2 -Ag NPs/SPE sensor. Similarly, we analyzed the repeatability, stability, and specificity of the BSA/Anti-PSMA/Ti 3 C 2 -Ag NPs/SPE sensor. The RSD of current response of the PSMA sensor is 0.73% (Fig. 5 A). After storage for 3, 6, 9, and 12 days, the current response decreased by 5.38%, 8.24%, 9.61%, and 9.90%, respectively. After nearly two weeks of storage, the current response of the BSA/Anti-PSMA/Ti 3 C 2 -Ag NPs/SPE sensor reached 90.10% of its original value (Fig. 5 B). The current response of the BSA/Anti-PSMA/Ti 3 C 2 -Ag NPs/SPE sensor with Albumin, glucose, and BSA was 5.01%, 1.34%, and 4.61%, respectively (Fig. 5 C). These results demonstrate that the BSA/Anti-PSMA/Ti 3 C 2 -Ag NPs/SPE sensor also exhibits excellent repeatability, stability, and specificity. 3.6 Clinical Validation with Human Serum Samples To validate clinical applicability of our platform, the dual-channel label-free EIs was employed for the simultaneous detection of PSA and PSMA in human serum samples. Following the acquisition of a baseline signal, the immunosensors were incubated with serum samples (diluted 1:10 in PBS) for 30 minutes. As illustrated in Fig. 6 , the subsequent formation of antibody-antigen immunocomplexes on the electrode surface induced a quantifiable attenuation in the redox peak current (Δi). The magnitude of Δi demonstrated a direct and robust correlation with the target analyte concentrations. For instance, Sample 1, associated with a lower pathological grade, exhibited a minimal Δi, whereas Sample 4, corresponding to a higher grade of disease severity, produced the most pronounced signal suppression. The intermediate Δi values observed for Samples 2 and 3 were quantitatively consistent with their respective pathological staging, thereby corroborating the platform's capacity for accurate clinical stratification. The precise quantification of PSA and PSMA is paramount for the diagnosis and prognostic monitoring of PCa. According to established clinical guidelines, serum concentrations exceeding 4 ng/mL for PSA [ 37 ] and 300 ng/mL for PSMA [ 38 ] are indicative of an elevated risk for PCa. To assess the clinical readiness of our platform, we analyzed human serum samples, quantifying PSA and PSMA via our established calibration models. First, to validate its analytical fidelity, the PSA concentrations measured by our biosensor were benchmarked against the gold-standard enzyme-linked immunosorbent assay (ELISA). The results in Table 1 demonstrate excellent concordance, with relative deviations ranging from − 6.2% to + 11.7%, thereby confirming the platform’s reliability and accuracy. However, the clinical utility of PSA is often compromised by its limited diagnostic specificity. Its concentration is frequently elevated in benign conditions such as age-related prostate enlargement, prostatic inflammation, and urinary tract infections, a well-documented limitation that lead to a high incidence of false-positive diagnoses and necessitates the integration of complementary biomarker [ 39 ]. PSMA, a transmembrane glycoprotein overexpressed in advanced PCa, is pivotal for its enhanced specificity for malignant transformation. Its expression level is a direct indicator of tumor aggressiveness and metastatic potential [ 40 ], making it particularly valuable for disease staging and progression monitoring. Critically, our biosensor’s diagnostic conclusions achieved complete concordance with the gold-standard findings, unequivocally affirming its clinical applicability. This result substantiates the superior diagnostic precision of our multiplexed approach over single-marker assays. By integrating rapid, low-cost analysis with minimal sample requirements, our platform is thus poised to become a powerful point-of-care testing (POCT) tool, particularly for resource-limited healthcare settings Table 1 Detection of PSA and PSMA in clinical samples using the dual-channel label-free EIs No. of clinical serum Pathology results PSA ELISA results (ng·mL − 1 ) PSA test current (µA) Average current (µA) Average concentration (ng·mL − 1 ) PSMA test current (µA) Average current (µA) Average concentration (ng·mL − 1 ) 1 Healthy 2.611 5.291 5.053 2.449 8.123 8.395 221.584 4.457 8.085 5.411 8.977 2 Prostate cancer 8.329 6.115 6.185 9.234 9.057 9.212 477.361 6.064 8.971 6.376 9.608 3 Prostate cancer 24.645 7.433 7.117 27.542 9.761 9.483 615.549 6.812 9.525 7.106 9.163 4 Prostate cancer 80.733 7.542 8.079 85.114 9.971 9.725 773.145 8.185 9.653 8.510 9.551 4 Conclusion A dual-channel label-free EI was developed to simultaneously detect PSA and PSMA. The platform was constructed using Ti 3 C 2 -Ag NPs nanocomposites immobilized onto n a screen-printed three-electrode system. Following a mild synthesis of the Ti 3 C 2 nanosheets, the Ti 3 C 2 -Ag NPs nanocomposites were prepared via a facile self-reduction process, wherein the incorporation of Ag NPs significantly enhanced the nanocomposites’ electrical conductivity. The resulting EIs demonstrated a wide linear response range of 0.1-1,000 ng·mL − 1 for both PSA and PSMA, with low sensitivities of 0.0036 µA·mL·ng − 1 and 0.0024 µA·mL·ng − 1 , respectively. The limits of detection were determined to be 0.045 ng·mL − 1 for PSA and 0.041 ng·mL − 1 for PSMA. Furthermore, the immunosensor exhibited excellent repeatability, long-term stability, and high specificity, as confirmed by rigorous experimental validation. In clinical validation studies using serum samples from healthy donors and prostate cancer patients, the platform successfully discriminated between the two cohorts, underscoring its potential for application in multiplexed clinical immunoassays. The innovative dual-channel architecture facilitates multiplexed biomarker quantification free from cross-interference, providing a cost-effective, rapid, and scalable platform for point-of-care diagnostics. This methodology presents a versatile and powerful framework for the parallel detection of diverse disease biomarkers, holds significant promise for advancing precision medicine through the design of tailored biosensor architectures. Declarations Author Contribution Song song Yang wrote the main manuscript text.Lu Han organized and coordinated this work.Wulin Xin and Heqing Cai prepared figures 6 and table.1.Kou Zhang, Xinyu Xue and Zhicheng Sun made their contribution to polishing and revising the text of this manuscript, especially in the reference part.Lei Wang offered blood samples of prostate cancer patients. Peng Liu provided the financial support.All authors reviewed the manuscript. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (No. 82274489 and No.22278037), 2024 Capital's Funds for Health Improvement and Research-Application of holographic three-dimensional imaging technology in partial nephrectomy of renal tumors (Number: SFH2024-2-6044). References Han L, Liu C-M, Dong S-L, Du C-X, Zhang X-Y, Li L-H, Wei Y (2017) Enhanced conductivity of rGO/Ag NPs composites for electrochemical immunoassay of prostate-specific antigen. 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Adv Sci 10(15):2206494 Corpetti M, Müller C, Beltran H, de Bono J, Theurillat J-P (2024) Prostate-Specific Membrane Antigen–Targeted Therapies for Prostate Cancer: Towards Improving Therapeutic Outcomes. Eur Urol 85(3):193–204 Additional Declarations No competing interests reported. Supplementary Files Surpportinginformation.docx Graphicalabstract.tif Cite Share Download PDF Status: Published Journal Publication published 16 Feb, 2026 Read the published version in Microchimica Acta → Version 1 posted Editorial decision: Revision requested 12 Sep, 2025 Reviews received at journal 11 Sep, 2025 Reviews received at journal 10 Sep, 2025 Reviews received at journal 08 Sep, 2025 Reviews received at journal 07 Sep, 2025 Reviews received at journal 04 Sep, 2025 Reviewers agreed at journal 04 Sep, 2025 Reviewers agreed at journal 04 Sep, 2025 Reviewers agreed at journal 04 Sep, 2025 Reviewers agreed at journal 03 Sep, 2025 Reviewers agreed at journal 03 Sep, 2025 Reviewers agreed at journal 03 Sep, 2025 Reviewers invited by journal 03 Sep, 2025 Editor assigned by journal 01 Sep, 2025 Submission checks completed at journal 31 Aug, 2025 First submitted to journal 27 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7470190","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":512224725,"identity":"4457b17c-a0cd-4489-9f4d-d22b995132d3","order_by":0,"name":"Song-Song Yang","email":"","orcid":"","institution":"Beijing Institute of Graphic Communication","correspondingAuthor":false,"prefix":"","firstName":"Song-Song","middleName":"","lastName":"Yang","suffix":""},{"id":512224726,"identity":"82804a35-668b-47b6-9610-36f3dece351c","order_by":1,"name":"Lu Han","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtklEQVRIiWNgGAWjYBACAwYGxgMMFRKkaWE4wHCGZC2MbaQ4zJz97IHDvPMs5Pnbjz9g+FHDIG9OSItlT17CYd5tEoYzzuQYMPYcYzDc2UDIYQdyDEBaEgwYchgYeBsYEgwOENJy/g1QyxygFv7nDxj/EqXlBsiWBqAWIGImzpYbbwwOzjkG9AuQcVgGyNhA2GE5hg/e1NTJ8/enP3z4psZGnqAtKAComJQ4HQWjYBSMglGAEwAA8gk+yvU4ZIQAAAAASUVORK5CYII=","orcid":"","institution":"Beijing Institute of Graphic Communication","correspondingAuthor":true,"prefix":"","firstName":"Lu","middleName":"","lastName":"Han","suffix":""},{"id":512224727,"identity":"ac7cc639-daae-4f92-ad09-e5e99ef9e278","order_by":2,"name":"Wu-Lin Xin","email":"","orcid":"","institution":"Beijing Institute of Graphic Communication","correspondingAuthor":false,"prefix":"","firstName":"Wu-Lin","middleName":"","lastName":"Xin","suffix":""},{"id":512224728,"identity":"bb53f427-8eed-429e-b985-2862f3f77da0","order_by":3,"name":"He-Qing 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09:38:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7470190/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7470190/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00604-026-07838-6","type":"published","date":"2026-02-16T15:59:08+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91073534,"identity":"cfbb6119-1249-4d2d-a26d-0b9ecc079284","added_by":"auto","created_at":"2025-09-11 10:59:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":12110298,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of Ti\u003csub\u003e3\u003c/sub\u003eAlC\u003csub\u003e2 \u003c/sub\u003e(A and B)\u003csub\u003e \u003c/sub\u003eof Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2 \u003c/sub\u003enanosheets\u003csub\u003e \u003c/sub\u003e(C and D)\u003c/p\u003e","description":"","filename":"fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7470190/v1/9a7da62c9acc251f1c9792a1.png"},{"id":91071194,"identity":"3eaf8ddd-1b1c-4ba6-8046-a6d04c067157","added_by":"auto","created_at":"2025-09-11 10:51:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":15360638,"visible":true,"origin":"","legend":"\u003cp\u003eUV-vis (A) and XRD (B) spectra of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e nanosheets and Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs nanocomposites; TEM images of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2 \u003c/sub\u003enanosheets (C) and Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs nanocomposites (E); Enlarged TEM of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs nanocomposites (D)\u003c/p\u003e","description":"","filename":"fig.21.png","url":"https://assets-eu.researchsquare.com/files/rs-7470190/v1/3d6eb288ef15572f8353d0e1.png"},{"id":91071186,"identity":"081478ce-2280-43f0-9d39-1d901577fac8","added_by":"auto","created_at":"2025-09-11 10:51:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":8173038,"visible":true,"origin":"","legend":"\u003cp\u003eThe conductivity (A) of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2 \u003c/sub\u003eand Ti\u003csub\u003e3\u003c/sub\u003eAlC\u003csub\u003e2\u003c/sub\u003e; CVs (B) and SWVs (C) of bare SPE, Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e/SPE and Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE; CVs (D) and peak currents (E) of SPEs modified by Ti3C2-Ag NPs with various concentrations\u003c/p\u003e","description":"","filename":"fig.31.png","url":"https://assets-eu.researchsquare.com/files/rs-7470190/v1/2142c8cb48d09f5bcdbdbdc8.png"},{"id":91073533,"identity":"4a59395b-2521-4cc9-8cd9-97a950ce5658","added_by":"auto","created_at":"2025-09-11 10:59:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":8977860,"visible":true,"origin":"","legend":"\u003cp\u003eCV (A) and EIS (B) curves of BSA/Anti-PSA/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE sensor; (C) Calibration and logarithmic calibration curves of the relationship between oxidation peak current changes (Δi) and PSA concentration of BSA/Anti-PSA/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE sensor ranging from 0.1 to 1,000 ng·mL\u003csup\u003e-1\u003c/sup\u003e; CV (D) and EIS (E) curves of BSA/Anti-PSMA/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE sensor; (F) Calibration and logarithmic calibration curves of the relationship between oxidation peak current changes (Δi) and PSMA concentration of BSA/Anti-PSMA/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE sensor ranging from 0.1 to 1,000 ng·mL\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-7470190/v1/09397df2afe886d1f44e17a6.png"},{"id":91075413,"identity":"2332cb0b-1237-4db7-bf0b-0c776bf1521a","added_by":"auto","created_at":"2025-09-11 11:07:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":11231409,"visible":true,"origin":"","legend":"\u003cp\u003eCV current responses of BSA/Anti-PSA/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE sensors and BSA/Anti-PSMA/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE sensors of different batches (A) and stored for different time intervals (B). (C) Response current responses of BSA/Anti-PSA/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE sensor and BSA/Anti-PSMA/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE sensors incubating with different interfering substances: (Ⅰ) 1,000 ng·mL\u003csup\u003e-1\u003c/sup\u003e PSA (or PSMA), (Ⅱ) 1,000 ng·mL\u003csup\u003e-1\u003c/sup\u003e PSA (or PSMA)+100 ng·mL\u003csup\u003e-1\u003c/sup\u003e Albumin, (Ⅲ) 1,000 ng·mL\u003csup\u003e-1\u003c/sup\u003e PSA (or PSMA)+100 ng·mL\u003csup\u003e-1\u003c/sup\u003e glucose, (Ⅳ) 1,000 ng·mL\u003csup\u003e-1\u003c/sup\u003e PSA (or PSMA)+100 ng·mL\u003csup\u003e-1\u003c/sup\u003e BSA.\u003c/p\u003e","description":"","filename":"fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-7470190/v1/13f80f74ff5d216fa7877b96.png"},{"id":91071193,"identity":"913edb8d-ad7e-47f2-a9db-be231c91ace4","added_by":"auto","created_at":"2025-09-11 10:51:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":11930373,"visible":true,"origin":"","legend":"\u003cp\u003eCV current responses of BSA/Anti-PSA/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE sensors (A) and BSA/Anti-PSMA/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE sensors (B) of different clinical serum samples.\u003c/p\u003e","description":"","filename":"fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-7470190/v1/3e0a987b0bd0528dd35d84ad.png"},{"id":103253071,"identity":"509bff44-9e23-4a59-80f6-5df3c85fee4c","added_by":"auto","created_at":"2026-02-23 16:17:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":57824474,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7470190/v1/239a68c5-ac5d-4407-b4d4-d3398ed5267f.pdf"},{"id":91071190,"identity":"3f624e13-d0e7-4351-a512-bc69f5723ea0","added_by":"auto","created_at":"2025-09-11 10:51:12","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1789258,"visible":true,"origin":"","legend":"","description":"","filename":"Surpportinginformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7470190/v1/3599717fa01a1e92435e9832.docx"},{"id":91071200,"identity":"596a90eb-4903-4c36-958b-0b9ada470489","added_by":"auto","created_at":"2025-09-11 10:51:13","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":9851112,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-7470190/v1/c842e4dd6d0c0583f9f9eb6e.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"An Integrated Electrochemical Platform Based on Ti 3 C 2 -Ag NPs and 3D- Printed Microfluidics for Simultaneous Detection of PSA and PSMA","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eLabel-free electrochemical immunosensors (EIs) have emerged as a transformative technology for cancer biomarker detection, offering unparalleled advantages in sensitivity, specificity, rapid response, cost-effectiveness, and user-friendliness [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Despite these merits, conventional single-channel EIs, which target individual biomarkers, suffer from high rates of false positives/negatives in clinical settings, limiting their diagnostic reliability [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. To address this critical gap, the development of multi-channel EIs has gained momentum, promising enhanced diagnostic accuracy, reduced analysis time, and lower operational costs through simultaneous multi-analyte detection. A typical multi-channel EI system integrates a reference electrode, an auxiliary electrode, and multiple working electrodes. However, signal interference (cross-talk) between adjacent working electrodes primarily caused by diffusion of electroactive species\u0026mdash;remains a significant technical hurdle [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. While prior strategies, such as minimizing electrode surface area [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and increasing inter-electrode spacing [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], mitigate cross-talk, they inadvertently compromise sensor performance by elevating the limit of detection (LOD) or reducing sensitivity. Thus, innovative approaches are urgently needed to resolve this trade-off without sacrificing analytical efficacy.\u003c/p\u003e\u003cp\u003eScreen-printed electrodes (SPEs), serving as the central component of label-free EIs, offer significant advantages, notably their cost-effectiveness, user-friendly operation, and scalability [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Advanced screen-printing techniques enable precise fabrication of disposable electrodes with micron-scale resolution. Furthermore, SPEs can be tailored to meet the specific requirements of various types of EIs, allowing for the creation of diverse patterns through layering techniques. Consequently, novel EIs can be developed with confidence by leveraging the versatility of SPEs [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The performance of these systems is critically dependent on electrode materials, because appropriate electrode materials can offer a substantial specific surface area for immobilizing a greater number of antibodies and superior electrical conductivity to facilitate electron transfer on the electrode surface [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Two-dimensional (2D) layered nanomaterials, including transition metal sulfides [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], black phosphorus [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], layer double hydroxide (LDH) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and MXenes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] have gained widespread use owing to their unique layered structures, large specific surface areas, ultra-thin nature, excellent physicochemical properties, and thickness-dependent band gaps [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Among these, MXenes offer exceptional versatility through tunable M/X element combinations and surface functionalization [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], exemplified by the metallic conductivity of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-MXene [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, their intrinsic 2D structures are prone to restacking, hindering electron transfer on the electrode surface of EIs and limiting their electrochemical performance [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Recent advances demonstrate that MXene nanocomposites, which composite conductive materials including noble metal nanoparticles (MNPs), carbon nanostructures, and conductive polymers via the numerous functional groups (-OH, -F, =O, etc.) on the surface, can overcome these limitations through synergistic interfacial interactions [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Among MNPs, gold nanoparticles (Au NPs) and silver nanoparticles (Ag NPs) are particularly versatile and widely employed in biosensing, catalysis, and medical imaging due to their nanoscale effects and excellent electrical conductivity [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Ag NPs, in particular, offer exceptional cost-effectiveness, making them the preferred choice for reducing the production costs of MXene-based nanocomposites.\u003c/p\u003e\u003cp\u003eHere, we address these challenges through a dual-pronged approach. Firstly, we leverage SPEs to fabricate a three-electrode system with dual working electrodes modified by Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs nanocomposites. This design amplifies electrochemical signals while maximizing antibody immobilization density. Secondly, we propose an innovative approach that combines microfluidic [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and stereolithography (SLA) 3D printing technologies to create polydimethylsiloxane (PDMS) microfluidic channels with spatially isolated reaction chambers to eliminate cross-talk between electrodes. This architecture ensures precise localization of prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) antibodies on distinct working electrodes, enabling simultaneous and interference-free detection. The LOD values achieved for the developed dual-channel label-free EIs were 0.045 ng\u0026middot;mL-1 for PSA and 0.041 ng\u0026middot;mL-1 for PSMA, displaying their high sensitivity, specificity, and low detection limits. Finally, the sensors were used for clinical detection and quantification of PSA and PSMA in serum samples, which exhibit consistent results compared with a specialized instrument, demonstrating this dual-channel electrochemical sensor has been validated as a reliable tool for early prostate cancer (PCa) diagnosis.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Materials\u003c/h2\u003e\u003cp\u003ePSA, PSMA, albumin, and lithium fluoride (LiF) were acquired from Sigma-Aldrich. PSA antibody and PSMA antibody were procured from Cell Signaling Technology. Aluminum titanium carbide (Ti\u003csub\u003e3\u003c/sub\u003eAlC\u003csub\u003e2\u003c/sub\u003e), bovine serum albumin (BSA), glucose, and phosphate-buffered saline (PBS) were purchased from Shanghai Macklin Biochemical Co., Ltd. Silver paste, carbon paste, and insulating ink were obtained from Jujo Chemical Co., Ltd. Polyethylene terephthalate (PET) film (100 \u0026micro;m) and PDMS were sourced from Beijing Chemical Plant. All other reagents were of analytical grade and employed without further purification. Clinical serum samples were obtained from Peking University Shougang Hospital (Beijing, China). Ethics Committee of Peking University Shougang Hospital approved the use of this material for research purposes, and informed consent was provided from all patients (approval number: 23587-0-01).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Synthesis of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e nanosheets\u003c/h2\u003e\u003cp\u003eAs shown in sheme.1A, layered Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-MXene was exfoliated via minimally intensive layer delamination [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Briefly, 1.6 g LiF was dissolved in 20 mL HCl (15 mL 37% HCl\u0026thinsp;+\u0026thinsp;5 mL H\u003csub\u003e2\u003c/sub\u003eO), followed by gradually add 1 g of Ti\u003csub\u003e3\u003c/sub\u003eAlC\u003csub\u003e2\u003c/sub\u003e and stir the reaction (38 \u0026deg; C, 48 h). The product was centrifuged (3,500\u0026ndash;8000 rpm, 10 min) and washed with ultra-pure water multiple times until the pH of the supernatant reached approximately 6. The bottle-green supernatant containing Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e nanosheets was collected and subsequently freeze-dried for further testing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Preparation of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs nanocomposites\u003c/h2\u003e\u003cp\u003eTi\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs nanocomposites were synthesized using a self-reduction method (Sheme.1A). Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e suspension (5 mL) was mixed with 5 mL H\u003csub\u003e2\u003c/sub\u003eO, followed by dropwise addition of 200 \u0026micro;L 0.1 M AgNO\u003csub\u003e3\u003c/sub\u003e under stirring at 25 \u0026ordm;C (1000 rpm, 1 h). Centrifuge (8,000 rpm, 10 min) and collect the supernatant to obtain the Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs nanocomposites for further characterization and application.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Characterization of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e and Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs nanocomposites\u003c/h2\u003e\u003cp\u003eThe structures of the as-prepared Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e nanosheets and Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs nanocomposites were characterized using Ultraviolet-visible (UV-vis, UV-2501PC, SHIMADZU, Japan), Fourier transform infrared (FTIR, IS-5, Thermo Scientific, USA), X-ray diffractometer (XRD, Bruker D8, Bruker AXS, Germany), and Raman spectroscopy (Xplora-BX51, HORIBA, France). The microstructural and morphological features of the as-prepared Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e nanosheets and Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs nanocomposites were examined using scanning electron microscopy (SEM, SU8020, HITACHI, Japan) and transmission electron microscopy (TEM, F20, FEI, Netherlands).The conductivity was measured using a four-point probe system (RTS-9, Guangzhou, China) and surface profiler (Dektak 150, Veeco, USA) and the specific operation is recorded in the supporting information.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Construction of the dual-channel label-free EIs\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eScheme.1\u003c/b\u003e Preparation route of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs nanocomposites and the dual-channel label-free EIs (A); Application of PCa diagnosis based on the dual-channel label-free EIs (B)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Electrochemical properties\u003c/h2\u003e\u003cp\u003eThe electrochemical performance of all samples was evaluated using an electrochemical workstation (AutoLab, Metrohm, Switzerland). Cyclic voltammetry (CV) and square wave voltammetry (SWV) were performed in 0.01 M PBS (pH 7.4) containing 5 mM K\u003csub\u003e3\u003c/sub\u003e[Fe(CN)\u003csub\u003e6\u003c/sub\u003e], 5 mM K\u003csub\u003e4\u003c/sub\u003e[Fe(CN)\u003csub\u003e6\u003c/sub\u003e], and 0.1 M KCl. CV tests were conducted in the potential range of -0.2 V to 0.6 V at a scanning rate of 100 mV\u0026middot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, while SWV tests were performed in the potential range of -100 mV to 600 mV at an amplitude of 20 mV.\u003c/p\u003e\u003cp\u003eIncubate (25\u0026deg; C, 3 h) 3 \u0026micro; L of PSA and PSMA diluted solutions of different concentrations (0.1\u0026ndash;1000 ng\u0026middot;mL⁻\u003csup\u003e1\u003c/sup\u003e) on the corresponding electrodes of dual-channel label-free EIs, wash multiple times, and analyze the immunosensor using CV and electrochemical impedance spectroscopy (EIS) to evaluate sensitivity, linear dynamic range, repeatability, stability, and specificity. The frequency range measured by EIS is 0.1 Hz to 1 MHz, with an amplitude of 120 mV.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Detection of PSA and PSMA in clinical samples\u003c/h2\u003e\u003cp\u003eTo assess clinical applicability, serum levels of PSA and PSMA were quantified in four anonymized human samples using the developed dual-channel label-free EIs. Briefly, serum samples from 1 healthy donor and 3 prostate cancer patients was diluted 1:10 in PBS. Samples (3 \u0026micro;L) were incubated on antigen-specific electrodes (37\u0026deg;C, 30 min), washed, and analyzed via CV. The specific operation is recorded in the supporting information.\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Results and Discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Characterization of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e nanosheets\u003c/h2\u003e\u003cp\u003eTo confirm the successful exfoliation of the \"Al\" layer from Ti\u003csub\u003e3\u003c/sub\u003eAlC\u003csub\u003e2\u003c/sub\u003e, UV-vis, FTIR, XRD, and Raman spectroscopy were employed to characterize the Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e nanosheets, the results of which were shown in Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The SEM images in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and B reveal the presence of a distinct layered structure in Ti\u003csub\u003e3\u003c/sub\u003eAlC\u003csub\u003e2\u003c/sub\u003e on silicon wafers, with the layers tightly adhering to each other. However, after undergoing MILD treatment, Ti\u003csub\u003e3\u003c/sub\u003eAlC\u003csub\u003e2\u003c/sub\u003e transforms into a highly thin, smooth, and flaky sheet structure, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and D. Additionally, the folding and stacking of these ultra-thin Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-MXene sheets are evident, further confirming the successful production of mono- or few-layered Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e. The significantly larger specific surface area of the ultra-thin Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e, compared to Ti\u003csub\u003e3\u003c/sub\u003eAlC\u003csub\u003e2\u003c/sub\u003e, facilitates the binding of Ag NPs, thereby enhancing the electrochemical performance of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-MXene.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Characterization of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs nanocomposites\u003c/h2\u003e\u003cp\u003eTo determine whether Ag NPs were effectively anchored onto Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e nanosheets, UV-vis and XRD and TEM analyses were conducted. The surface plasmon resonance (SPR) characteristic peak of Ag NPs at 425 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and the (111) crystal face peak of Ag NPs at 39\u0026deg; (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) were both observed in Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs nanocomposites, indicating successful integration of Ag NPs with Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e. A typical TEM image of mono- or few-layered Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-MXene with gauze-like sheets is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC. After sufficient reaction with AgNO\u003csub\u003e3\u003c/sub\u003e, a large number of uniformly distributed Ag NPs with small particle size were observed on the surface of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e sheets (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), providing further evidence of successful preparation of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs nanocomposites. This can be attributed to the residual\u0026thinsp;=\u0026thinsp;O, -OH, and -F moieties on the surface of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e nanosheets, which provided a sufficient number of chemically active sites for deposition of Ag NPs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Electrochemical properties of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs nanocomposites\u003c/h2\u003e\u003cp\u003eTo investigate the impact of \"Al\" exfoliation on the conductivity of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e, we measured the electrical conductivity of Ti\u003csub\u003e3\u003c/sub\u003eAlC\u003csub\u003e2\u003c/sub\u003e and Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e using a four-point probe resistivity measurement system. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, the conductivity of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e was found to be 3989 S\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which is 15 times higher than that of Ti\u003csub\u003e3\u003c/sub\u003eAlC\u003csub\u003e2\u003c/sub\u003e (257 S\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and consistent with previously reported literature [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo determine whether the integration of Ag NPs with Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e could enhance the electrical conductivity of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e, we immobilized as-synthesized Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e nanosheets and Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs nanocomposites onto SPEs and measured their CV and SWV curves. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e/SPE exhibited a larger redox peak current and a smaller potential difference than bare SPE due to the accelerated electron transfer effect by Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e nanosheets. The redox peak current of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE continued to increase significantly, and the potential difference of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE further decreased, indicating that the incorporation of Ag NPs onto Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e nanosheets could further enhance the electron transfer on Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e nanosheets. Furthermore, SWV curves (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) demonstrated a significantly higher peak current for Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE compared to bare SPE and Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e/SPE. The peak current of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE was 2.38 times higher than that of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e/SPE, suggesting that Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs can effectively enhance the electron transfer rate on the surface of SPE, which was consistent with the CV results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). These results indicate that the electrical conductivity of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e can be remarkably enhanced by the insertion of Ag NPs, making it a promising electrode material for the construction of label-free EIs. To determine the optimal amount of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs for electrode modification, various concentrations (50, 100, 150, 200, 300, 400, and 500 \u0026micro;g\u0026middot;mL-1) of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs solution (3 \u0026micro;L) were deposited onto SPE(a) and SPE(b) electrodes, and their corresponding CV curves were recorded. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and E, the peak current exhibited a continuous increase with increasing concentrations of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs from 0 to 150 \u0026micro;g\u0026middot;mL-1, accompanied by a decrease in the peak potential difference. These observations indicate an enhanced electron transfer rate. However, beyond the concentration of 150 \u0026micro;g\u0026middot;mL-1, the peak current gradually decreased, and the potential difference increased. Consequently, the optimal modification amount of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs onto SPEs was determined to be 150 \u0026micro;g\u0026middot;mL-1.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Electrochemical analysis of dual-channel label-free EIs\u003c/h2\u003e\u003cp\u003eA dual-channel label-free EI for the detection of PSA and PSMA was developed using Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs nanocomposites as highly efficient substrate materials. The changes in electrochemical behavior of the BSA/Anti-PSA/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE sensor and BSA/Anti-PSMA/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE sensor at each assembly step were investigated using ferricyanide as an indicator, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and D. It can be observed that the redox peak currents of the SPEs were significantly enhanced, and the redox potential difference was remarkably reduced after the modification with Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs nanocomposites. Subsequently, a gradual decrease in the peak current was observed after the sequential immobilization of Anti-PSA (or Anti-PSMA), BSA, and the corresponding PSA (or PSMA), indicating the successful immobilization of these molecules onto the dual-channel label-free EI and the inhibition of electron transfer on the SPEs surfaces [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The incorporation of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs amplified the response of the fabricated label-free EIs, leading to an increase in the peak current, as Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs serve as efficient conductors to enhance the signal.\u003c/p\u003e\u003cp\u003eEIS is a powerful technique for monitoring changes in the surface properties of modified electrodes. The EIS typically consists of a semicircular region corresponding to the electron transfer process at higher frequencies and a linear region corresponding to the electron diffusion process at lower frequencies. The diameter of the semicircle represents the electron-transfer resistance (R\u003csub\u003eet\u003c/sub\u003e) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and E, the corresponding EIS spectra of the developed BSA/Anti-PSA/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE sensor and BSA/Anti-PSMA/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE sensor at different stages are presented. The bare SPE exhibited the highest resistance. However, upon modification with Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs, a significant decrease in the R\u003csub\u003eet\u003c/sub\u003e value was observed, indicating enhanced electron transfer facilitated by Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Subsequently, a gradual increase in R\u003csub\u003eet\u003c/sub\u003e values was observed after sequential modification of Anti-PSA (or Anti-PSMA), BSA, and PSA (or PSMA) onto SPE(a) (or SPE(b)), which can be attributed to the successful immobilization of these protein molecules. The formation of the antibody-antigen immunocomplex on the electrode interface hindered charge transfer [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. These results further confirm the successful fabrication of the dual-channel label-free EIs, which can be utilized for the detection of PSA and PSMA.\u003c/p\u003e\u003cp\u003eThe LOD values for the label-free EIs were determined by analyzing a series of antigens with varying concentrations [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The calibration plot and logarithmic calibration curve for PSA and PSMA detection under optimal experimental conditions are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and F. The current response exhibited a decrease with increasing concentrations of PSA and PSMA, and a linear relationship was observed between the change in current (Δi) and the logarithm of PSA and PSMA concentrations within the range of 0.1 to 1,000 ng\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The linear equation for the BSA/Anti-PSA/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE sensor was determined as 6.25291\u0026thinsp;+\u0026thinsp;1.96397x, with a correlation coefficient of 0.99736. Similarly, the linear equation for the BSA/Anti-PSMA/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE sensor was found to be 5.09592\u0026thinsp;+\u0026thinsp;2.45162x, with a correlation coefficient of 0.99673. The LOD values of the sensor were calculated using the formula X\u003csub\u003eb\u003c/sub\u003e+KS\u003csub\u003eb\u003c/sub\u003e=algC\u003csub\u003eL\u003c/sub\u003e+b, where X\u003csub\u003eb\u003c/sub\u003e represents the average peak current change value of the bare electrode, S\u003csub\u003eb\u003c/sub\u003e is the standard deviation of the peak current change value of the bare electrode, K is the signal-to-noise ratio, and C\u003csub\u003eL\u003c/sub\u003e is the LOD of the sensor [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Consequently, the LOD of the dual-channel label-free EIs for PSA was determined to be 0.045 ng\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, with a linear detection range of 0.1-1,000 ng\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and a sensitivity of 0.0036 \u0026micro;A\u0026middot;mL\u0026middot;ng\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at a signal-to-noise ratio of 3. For PSMA, the LOD of the dual-channel label-free EIs was calculated as 0.041 ng\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, with a linear detection range of 0.1-1,000 ng\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and a sensitivity of 0.0024 \u0026micro;A\u0026middot;mL\u0026middot;ng\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at a signal-to-noise ratio of 3. Importantly, the serum PSA concentrations of both normal individuals (4 ng\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and prostate cancer patients (10 ng\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) fall within the linear range of the dual-channel label-free Eis [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Therefore, this study presents a novel method for simultaneous detection of PSA and PSMA concentrations, highlighting the potential clinical application of the developed dual-channel label-free EIs [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e presents a comparative analysis of proposed label-free EIs designed for the detection of PSA or PSMA. Previous studies have primarily focused on detecting either PSA or PSMA alone, limiting their applicability. In contrast, the EIs developed in this study enables the simultaneous detection of both PSA and PSMA at a lower LOD and a wide detection range. This advancement significantly enhances the accuracy of clinical analysis. While an electrochemical sensor for PSA and PSMA detection has been reported previously [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], its detection ranges is limited, and the LOD and detection ranges for PSA and PSMA are not aligned due to interference from different biomarker signals. In contrast, the label-free EIs in this work not only circumvent detection errors caused by signal crosstalk from the dual-channel working electrode, but also offer a broader detection range. More importantly, the EIs demonstrate a sensitivity of 0.0036 \u0026micro;A\u0026middot;mL\u0026middot;ng\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 0.0024 \u0026micro;A\u0026middot;mL\u0026middot;ng\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at the picomolar level for the detection of PSA and PSMA, respectively, across a reasonably broad concentration range. This capability enables rapid cancer diagnosis and holds promise for diverse biomedical applications.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Repeatability, stability and specificity of the dual-channel label-free EIs\u003c/h2\u003e\u003cp\u003eRepeatability is a crucial factor for biosensors, and it is important to verify the reliability of our developed label-free EIs. To this end, we randomly selected five samples from four different batches and performed CV tests [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, the current response of the PSA sensor exhibited a relative standard deviation (RSD) of 1.83%, indicating excellent repeatability. Stability is another essential characteristic of biosensors. To evaluate the stability of the BSA/Anti-PSA/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE sensor [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], we stored the sensors at 4 ℃ and examined their current responses every three days. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB illustrates that the BSA/Anti-PSA/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE sensor displayed a decrease in current response over time. After 3, 6, 9, and 12 days of storage, the current response of the sensor decreased by 6.78%, 14.18%, 14.68%, and 16.25%, respectively. After nearly two weeks of storage, the current response of the PSA sensor reached 83.75% of its original value, indicating good long-term stability of the BSA/Anti-PSA/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE sensor. Furthermore, to investigate the specificity of the BSA/Anti-PSA/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE sensor, we incubated the sensor with PSA in the presence of interfering agents, including Albumin, glucose, and BSA [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, the average current responses of the sensors exposed to interfering agents (Albumin 5.57%, glucose 2.42%, and BSA 2.77%) were within 4% of the immunosensor without interference, demonstrating the outstanding specificity of the BSA/Anti-PSA/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE sensor.\u003c/p\u003e\u003cp\u003eSimilarly, we analyzed the repeatability, stability, and specificity of the BSA/Anti-PSMA/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE sensor. The RSD of current response of the PSMA sensor is 0.73% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). After storage for 3, 6, 9, and 12 days, the current response decreased by 5.38%, 8.24%, 9.61%, and 9.90%, respectively. After nearly two weeks of storage, the current response of the BSA/Anti-PSMA/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE sensor reached 90.10% of its original value (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The current response of the BSA/Anti-PSMA/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE sensor with Albumin, glucose, and BSA was 5.01%, 1.34%, and 4.61%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These results demonstrate that the BSA/Anti-PSMA/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs/SPE sensor also exhibits excellent repeatability, stability, and specificity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Clinical Validation with Human Serum Samples\u003c/h2\u003e\u003cp\u003eTo validate clinical applicability of our platform, the dual-channel label-free EIs was employed for the simultaneous detection of PSA and PSMA in human serum samples. Following the acquisition of a baseline signal, the immunosensors were incubated with serum samples (diluted 1:10 in PBS) for 30 minutes. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, the subsequent formation of antibody-antigen immunocomplexes on the electrode surface induced a quantifiable attenuation in the redox peak current (Δi). The magnitude of Δi demonstrated a direct and robust correlation with the target analyte concentrations. For instance, Sample 1, associated with a lower pathological grade, exhibited a minimal Δi, whereas Sample 4, corresponding to a higher grade of disease severity, produced the most pronounced signal suppression. The intermediate Δi values observed for Samples 2 and 3 were quantitatively consistent with their respective pathological staging, thereby corroborating the platform's capacity for accurate clinical stratification.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe precise quantification of PSA and PSMA is paramount for the diagnosis and prognostic monitoring of PCa. According to established clinical guidelines, serum concentrations exceeding 4 ng/mL for PSA [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] and 300 ng/mL for PSMA [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] are indicative of an elevated risk for PCa. To assess the clinical readiness of our platform, we analyzed human serum samples, quantifying PSA and PSMA via our established calibration models. First, to validate its analytical fidelity, the PSA concentrations measured by our biosensor were benchmarked against the gold-standard enzyme-linked immunosorbent assay (ELISA). The results in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e demonstrate excellent concordance, with relative deviations ranging from \u0026minus;\u0026thinsp;6.2% to +\u0026thinsp;11.7%, thereby confirming the platform\u0026rsquo;s reliability and accuracy.\u003c/p\u003e\u003cp\u003eHowever, the clinical utility of PSA is often compromised by its limited diagnostic specificity. Its concentration is frequently elevated in benign conditions such as age-related prostate enlargement, prostatic inflammation, and urinary tract infections, a well-documented limitation that lead to a high incidence of false-positive diagnoses and necessitates the integration of complementary biomarker [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. PSMA, a transmembrane glycoprotein overexpressed in advanced PCa, is pivotal for its enhanced specificity for malignant transformation. Its expression level is a direct indicator of tumor aggressiveness and metastatic potential [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], making it particularly valuable for disease staging and progression monitoring. Critically, our biosensor\u0026rsquo;s diagnostic conclusions achieved complete concordance with the gold-standard findings, unequivocally affirming its clinical applicability. This result substantiates the superior diagnostic precision of our multiplexed approach over single-marker assays. By integrating rapid, low-cost analysis with minimal sample requirements, our platform is thus poised to become a powerful point-of-care testing (POCT) tool, particularly for resource-limited healthcare settings\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDetection of PSA and PSMA in clinical samples using the dual-channel label-free EIs\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNo. of clinical serum\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePathology results\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePSA ELISA results (ng\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePSA test current\u003c/p\u003e\u003cp\u003e(\u0026micro;A)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAverage current\u003c/p\u003e\u003cp\u003e(\u0026micro;A)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAverage concentration\u003c/p\u003e\u003cp\u003e(ng\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePSMA test current\u003c/p\u003e\u003cp\u003e(\u0026micro;A)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eAverage current\u003c/p\u003e\u003cp\u003e(\u0026micro;A)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eAverage concentration\u003c/p\u003e\u003cp\u003e(ng\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eHealthy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e2.611\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5.291\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e5.053\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e2.449\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e8.123\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e8.395\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e221.584\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4.457\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e8.085\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5.411\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e8.977\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eProstate\u003c/p\u003e\u003cp\u003ecancer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e8.329\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6.115\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e6.185\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e9.234\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e9.057\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e9.212\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e477.361\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6.064\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e8.971\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6.376\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e9.608\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eProstate\u003c/p\u003e\u003cp\u003ecancer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e24.645\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e7.433\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e7.117\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e27.542\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e9.761\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e9.483\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e615.549\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6.812\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e9.525\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e7.106\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e9.163\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eProstate\u003c/p\u003e\u003cp\u003ecancer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e80.733\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e7.542\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e8.079\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e85.114\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e9.971\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e9.725\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e773.145\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8.185\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e9.653\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8.510\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e9.551\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Conclusion","content":"\u003cp\u003eA dual-channel label-free EI was developed to simultaneously detect PSA and PSMA. The platform was constructed using Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs nanocomposites immobilized onto n a screen-printed three-electrode system. Following a mild synthesis of the Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e nanosheets, the Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs nanocomposites were prepared via a facile self-reduction process, wherein the incorporation of Ag NPs significantly enhanced the nanocomposites\u0026rsquo; electrical conductivity. The resulting EIs demonstrated a wide linear response range of 0.1-1,000 ng\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for both PSA and PSMA, with low sensitivities of 0.0036 \u0026micro;A\u0026middot;mL\u0026middot;ng\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 0.0024 \u0026micro;A\u0026middot;mL\u0026middot;ng\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. The limits of detection were determined to be 0.045 ng\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for PSA and 0.041 ng\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for PSMA. Furthermore, the immunosensor exhibited excellent repeatability, long-term stability, and high specificity, as confirmed by rigorous experimental validation. In clinical validation studies using serum samples from healthy donors and prostate cancer patients, the platform successfully discriminated between the two cohorts, underscoring its potential for application in multiplexed clinical immunoassays. The innovative dual-channel architecture facilitates multiplexed biomarker quantification free from cross-interference, providing a cost-effective, rapid, and scalable platform for point-of-care diagnostics. This methodology presents a versatile and powerful framework for the parallel detection of diverse disease biomarkers, holds significant promise for advancing precision medicine through the design of tailored biosensor architectures.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSong song Yang wrote the main manuscript text.Lu Han organized and coordinated this work.Wulin Xin and Heqing Cai prepared figures 6 and table.1.Kou Zhang, Xinyu Xue and Zhicheng Sun made their contribution to polishing and revising the text of this manuscript, especially in the reference part.Lei Wang offered blood samples of prostate cancer patients. Peng Liu provided the financial support.All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eThis work was financially supported by the National Natural Science Foundation of China (No. 82274489 and No.22278037), 2024 Capital's Funds for Health Improvement and Research-Application of holographic three-dimensional imaging technology in partial nephrectomy of renal tumors (Number: SFH2024-2-6044).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHan L, Liu C-M, Dong S-L, Du C-X, Zhang X-Y, Li L-H, Wei Y (2017) Enhanced conductivity of rGO/Ag NPs composites for electrochemical immunoassay of prostate-specific antigen. 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Polym Adv Technol 33(6):1967\u0026ndash;1977\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXie S, Fei X, Wang J, Zhu Y-C, Liu J, Du X, Liu X, Dong L, Zhu Y, Pan J, Dong B, Sha J, Luo Y, Sun W, Xue W (2023) Engineering the MoS2/MXene Heterostructure for Precise and Noninvasive Diagnosis of Prostate Cancer with Clinical Specimens. Adv Sci 10(15):2206494\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCorpetti M, M\u0026uuml;ller C, Beltran H, de Bono J, Theurillat J-P (2024) Prostate-Specific Membrane Antigen\u0026ndash;Targeted Therapies for Prostate Cancer: Towards Improving Therapeutic Outcomes. Eur Urol 85(3):193\u0026ndash;204\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Dual-channel electrochemical immunosensors, Multiplexed detection, Ti3C2-Ag NPs nanocomposites, Screen-printed electrodes","lastPublishedDoi":"10.21203/rs.3.rs-7470190/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7470190/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe multiplexed detection of tumor biomarkers represents a transformative strategy to improve diagnostic accuracy in oncology. A significant to the clinical translation of multiplexed label-free electrochemical immunosensors (EIs) is the issue of analytical reliability, which is frequently compromised by signal crosstalk and insufficient sensitivity. In this work, we designed a 3D-printed, reconfigurable microwell array through a combination of screen-printing, stereolithography (SLA) 3D printing, and microfluidic technologies for interference-free multiplexed detection. To achieve high sensitivity, the electrode surfaces were modified with a novel Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs nanocomposite that significantly enhances charge transfer kinetics by preventing Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e nanosheet aggregation through modulation of Ag NPs interlayer spacing. This integrated platform was validated through the simultaneous quantification of two critical prostate cancer biomarkers, prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA). The immunosensor demonstrated both the complete elimination of signal cross-talk and excellent analytical performance, including a wide linear range (0.1\u0026ndash;1,000 ng\u0026middot;mL⁻\u0026sup1;), low sensitivities (0.0036 \u0026micro;A\u0026middot;mL\u0026middot;ng\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for PSA and 0.0024 \u0026micro;A\u0026middot;mL\u0026middot;ng\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for PSMA), and low limits of detection (0.045 ng\u0026middot;mL⁻\u0026sup1; for PSA and 0.041 ng\u0026middot;mL⁻\u0026sup1; for PSMA). Furthermore, this device exhibited exceptional repeatability, stability, and specificity. Clinical validation using human serum samples exhibited strong concordance with clinical reference methods, enabling precise discrimination between prostate cancer patients and healthy controls. Consequently, the proposed dual-channel label-free EI, based on Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e-Ag NPs nanocomposites, holds substantial promise for clinical diagnostic applications, with potential for expansion to the ultrasensitive detection of other disease-related biomarkers.\u003c/p\u003e","manuscriptTitle":"An Integrated Electrochemical Platform Based on Ti 3 C 2 -Ag NPs and 3D- Printed Microfluidics for Simultaneous Detection of PSA and PSMA","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-11 10:51:07","doi":"10.21203/rs.3.rs-7470190/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-12T14:54:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-11T16:31:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-10T12:42:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-09T03:52:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-07T18:39:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-04T21:07:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"208268801633213191172095439625318036559","date":"2025-09-04T11:13:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"125278184082161519549517707566109302411","date":"2025-09-04T09:53:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"292841921732222392458988743256534461007","date":"2025-09-04T07:22:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"287034724995240675905214570298135132717","date":"2025-09-04T00:34:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"192183871914444627655441286405038125865","date":"2025-09-03T17:56:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"285361043181491174841497561060476745912","date":"2025-09-03T17:06:49+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-03T16:50:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-01T11:32:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-31T23:51:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microchimica Acta","date":"2025-08-27T09:25:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0419a639-25a7-4658-b0ea-3f985f06b5a2","owner":[],"postedDate":"September 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-23T16:15:57+00:00","versionOfRecord":{"articleIdentity":"rs-7470190","link":"https://doi.org/10.1007/s00604-026-07838-6","journal":{"identity":"microchimica-acta","isVorOnly":false,"title":"Microchimica Acta"},"publishedOn":"2026-02-16 15:59:08","publishedOnDateReadable":"February 16th, 2026"},"versionCreatedAt":"2025-09-11 10:51:07","video":"","vorDoi":"10.1007/s00604-026-07838-6","vorDoiUrl":"https://doi.org/10.1007/s00604-026-07838-6","workflowStages":[]},"version":"v1","identity":"rs-7470190","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7470190","identity":"rs-7470190","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}