Fabrication of Aptasensor for Monitoring cancer biomarkers: Detection of Lung Specific Biomarkers Using Chemical Converted Graphene

Fabrication of Aptasensor for Monitoring cancer biomarkers: Detection of Lung Specific Biomarkers Using Chemical Converted Graphene | 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 Fabrication of Aptasensor for Monitoring cancer biomarkers: Detection of Lung Specific Biomarkers Using Chemical Converted Graphene Jiqun Geng, Xiansong Yang, Ye Wang, Jun Che, Minwei Gu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6166131/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Sep, 2025 Read the published version in Chemical Papers → Version 1 posted 5 You are reading this latest preprint version Abstract This work was focused on development a stable, selective and sensitive electrochemical aptasensor based on 3,4,9,10-perylene tetracarboxylic acid -functionalized chemical converted graphene (PTCA/CCG) modified electrode for monitoring neuron specific enolase (NSE), lung-specific biomarkers. The electrochemical aptasensor was relied on the purified NSE aptamer immobilized on PTCA/CCG modified electrode. The PTCA/CCG promoted both the immobilization of the aptamer and the electrochemical signal. The aptasensor exhibited a low limit of detection (0.008 ng/mL), broad linear range (10 − 3 to 10 3 ng/mL), remarkable specificity, and excellent repeatability and long-term stability, maintaining initial resistance at 96.57% after 30 days. Results exhibited that the recoveries ranging from 98.00–99.00% and relative standard deviation less than 4.08%, demonstrating the acceptable precision and reliability of the fabricated aptadensor and confirming the aptasensor's performance and efficiency in complex biological fluids and clinical applications. Lung Cancer Biomarker Neuron Specific Enolase Electrochemical Aptasensor Chemical Converted Graphene Stability Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction When cells in a particular area of the body multiply and reproduce excessively, it can lead to cancer. The malignant cells have the ability to spread and obliterate nearby healthy tissue, including organs (Alshammary and Al-Attar 2024 ). Therefore, the cancer is one of the most important health challenges worldwide, emphasizing the critical need for advancements in treatment, medication, and early diagnosis. By concentrating on identifying people with symptoms as soon as possible, early cancer detection typically improves the likelihood of a successful course of therapy. Recognition and description of cancer cells are greatly aided by a variety of cancer diagnostic techniques, such as laboratory testing, diagnostic imaging, endoscopic examinations, genetic testing, and tumor biopsies (Kohaar et al. 2024 ). To more reliably and consistently identify biomarkers for patient treatment, prognosis, medical facilities, and societal well-being, there is a rising need for the creation of more effective techniques that make use of bioinformatics, the internet, and molecular biology. By reducing needless procedures and allocating healthcare resources as efficiently as possible, biomarkers can improve the precision of cancer detection and treatment and increase economic efficiency (Ahmad et al. 2024 ). A cancer biomarker might be either a biological molecule secreted by the tumor itself in bodily fluids such as blood or tissues which are the specific signs of a normal or abnormal process, or of a condition or disease such as cancer (Zakari et al. 2024 ). Specifically, lung cancer, one of the leading causes of cancer-related deaths, has its own set of biomarkers that these included pro-gastrin-releasing peptides(ProGRP), Squamous cells carcinoma-associated antigen(SCC), carcinoembryonic antigen (CEA), cytokeratin-19 fragment(CYFRA), and sialyl Lewis-X antigen(SLX) and neuron-specific enolase (NSE) (Mizuno et al. 2021 ). Between them, studied have been indicated that NSE is as an important biomarker for lung cancer, demonstrating the vital of its detection and patients having suspected lung cancer frequently have it measured (Huang et al. 2022 ; Kim et al. 2021 ). Aptamers are now biocompatible tools that can be utilized to find protein biomarkers. Short, single-stranded oligonucleotides called aptamers (DNA or RNA) fold into tertiary structures to attach to targets with great affinity and selectivity (Lingling Wu et al. 2021 ). Aptamers are used as identification elements in aptamer-based biosensors, or aptasensors, to detect a target, which is frequently connected to a readable signal. Aptamers-based probes are essential for precisely targeting cancer biomarkers (Kulabhusan et al. 2023 ). Aptamer-cancer biomarker affinity becomes crucial for targeted therapy, prognosis tracking, and diagnosis. Aptasensors' improved stability, adaptability, and capacity for regeneration for reusable biosensors are their primary advantages over immunosensors (Arshavsky-Graham et al. 2022 ). According to studies, aptasensors have a noticeably longer shelf life than immunosensors and may be kept in dry settings and for a while without losing their sensitivity (Al Mamun et al. 2021 ; Arshavsky-Graham et al. 2020 ; Arshavsky‐Graham et al. 2022). Electrochemical aptasensors are unique among the several aptasensor kinds due to their quick reaction, affordability, and simplicity of downsizing (Radi 2011 ). By utilizing the high signal amplification, distinct characteristics of different nanomaterials, and highly selective aptamer-target interactions, nanomaterial-based aptasensors are able to detect a variety of analytes of interest (Kurup et al. 2022 ). Due to their enormous surface area and remarkable conductivity, nanostructures like graphene oxide (GO) can sustain substantial biomolecule loading for excellent detection sensitivity (Amiri et al. 2021 ). Functionalized chemically converted graphene (CCG) offers additional benefits over traditional graphene oxide such as functionalization capabilities and improved stability (Bai et al. 2011 ; Bottari et al. 2017 ). The functionalized graphene sheets can be further altered by various chemical reactions, such as amidation, surface-initiated polymerization, which and reduction of metal ions, and they show better chemical and thermal stabilities than GO (Ismail et al. 2021 ). In addition to being electrically conductive, the CCG nanosheets exhibit superior solvent dispersibility and processability (Kuila et al. 2012 ). The incorporation of 3,4,9,10-perylene-tetracarboxylic-acid(PTCA) into CCG further improves the electrochemical properties of the aptasensor by providing better signal amplification and facilitating the electron transfer rate (Feng et al. 2011 ). The synergetic effect of PTCA and CCG (PTCA/CCG) leads to a highly effective sensing platform for monitoring cancer biomarkers. The creation of a innovative electrochemical-aptasensor based on a modified PTCA/CCG electrode for the monitoring of lung-specific biomarkers for NSE was the main goal of this work. The selection of these materials is based on their exceptional properties and the potential to promote aptasensor performance. This research aims to provide a novel and sensitive platform for the early diagnosis of lung cancer, contributing to low cost, better diagnostic tools and enhanced patient outcomes. 2. Experiments 2.1. Materials We bought electrodes made of glassy carbon (GCE, 5mm diameter) from Tokai Electrode Mfg. Co., Japan Alumina powder (1.0, 0.3, and 0.05 µm) was obtained from Buehler, USA. Ultrapage purified aptamer for NSE (5’- biotin-TEG-TCACACACGGACCTCTC CTACATTAATTGCGCATTTCGTT-3’) was ordered from Sangon Biotechnology Co. Recombinant human carcinoembryonic antigen (CEA) was purchased from Sangon Biotech, China. Alphafetoprotein (AFP) antigen and recombinant neuron specific enolase (NSE) protein were provided from Shanghai LincBio Science Co. Ltd., China. 1-ethyl-3- (3-dimethylaminopropyl)-carbodimide hydrochloride (EDC) was sourced from Pierce Biotechnology, Inc. (Rockford, USA). Alkaline phosphatase (ALP) was obtained from BBI Enzymes (Wisconsin, USA). N-Hydroxysuccinimide(NHS), bovine serum albumin(BSA), ammonia aqueous solution (25 wt%), hydrazine solution, sodium hydroxide (NaOH), MES buffer, Sigma-Aldrich (St. Louis, MO, USA) provided the tris buffer, potassium chloride (KCl), magnesium chloride (MgCl2), phosphate-buffered saline (PBS), ferricyanide of potassium ([Fe(CN)6] 3− ), and potassium-ferrocyanide([Fe(CN)6] 4− ). Ethanol and acetone came from Merck in Germany. 3,4,9,10-Thermo Scientific Chemicals in the United States provided the ethylene tetracarboxylic dianhydride (PTCDA). A Milli-Q purification equipment was used in the lab to create deionized water. 2.2. Instrumentation Dialysis membrane (Spectrapro membrane tubing, Spectrum Labs, CA, USA; MWCO 6–8 kDa). An EQUINOX-55 FTIR spectrometer was used to acquire FT-IR spectra. To characterize the synthesized nanomaterial, X-ray diffraction (XRD, X'Pert PRO, PANalytical), transmission electron microscopy (TEM, Tecnai G2 F20, FEI), scanning electron microscopy (SEM, JSM-7100F, JEOL Ltd., Japan), and X-ray photoelectron spectra (XPS) were employed. The actual samples were analyzed using the Human Neuron specific Enolase enzyme-linked immunosorbent test (ELISA) Kit (ab217778, Abcam). 2.3. Preparation of CCG and PTCA/CCG CCG was prepared according to the procedure reported by Li et al. (Dan Li et al. 2008 ). Briefly, ammonia aquatic solution (80 µL, 25 wt%) and hydrazine solution (5 µL) were added into the suspension of the GO (5 mL, 1 mg/mL). After 8 minutes magnetic stirring, the vial was placed in a water bath (90°C) for 60 minutes under vigorous stirring. For preparation of the PTCA/CCG, this procedure was conducted using mixture of ammonia aquatic solution, hydrazine solution, suspension of the GO and PTCA. PTCA was formed by hydrolyzing PTCAD (0.3 M) in sodium hydroxide (100 mL, 1M). a mixture of 3.92 g of PTCAD and NaOH was refluxed for 4 hours to obtain yellow-green solution (Sun et al. 1999 ). Following centrifugation, the solid was recovered and vacuum-dried at 37°C. A mixture of GO suspension (5 mL, 1 mg/mL) and PTCA powder (20 mg) was prepared by adding water to a final volume of 100 mL. After 30 minutes ultrasonication, the mixture was vigorously stirred at 40°C overnight. After that, hydrazine solution (5 µL) and ammonia solution (80 µL, 25 wt%) were added to the mixture. The resulted mixture was then heated at 90°C for 60 minutes under magnetic stirring. Next, the product was dialyzed using dialysis membrane for 3 days; dialysis water was changed 3 times daily. Finally, the precipitate was removed to reach the stable homogeneous solution. 2.4. Modification of Electrodes with Aptamer, CCG, and PTCA/CCG Before modification GCEs, GCEs were polished sequentially with 1.0, 0.3, and 0.05 µm Alumina powder, 5 minutes for each powder. Then, GCEs were sonicated for 10 minutes each in water, acetone and ethanol, and then dried under a directed stream of high-purity nitrogen. For modification, the PTCA/CCG mixture was mixed by a nafion solution (0.1%) in a 4:1 volume ratio to improve the affinity of the composite with the GCE. Then, PTCA/CCG-nafion solution (10 µL) was drop-casted onto the GCE and dried at 37°C. The carboxyl groups were then activated for 60 minutes by applying an activation solution that contained NHS (0.2 M) and EDC (0.1 M) into MES buffer (0.1 M). After that, the electrode surface was rinsed with 10 mM Tris buffer (pH = 7.4). The electrode surface was then drop-cast with aptamer (10 µL, 0.1 mM) containing a terminal amine group, and it was incubated for three hours. Afterwards, the modified electrode surface were washed with buffer and then hybridized by 1 mM of aptamer in solution (pH = 7.4) containing Tris buffer (10 mM), MgCl 2 (2.5 mM), KCl (140 mM) for 60 minutes. To block the remainder of bonding sites on the PTCA/CCG surface, the modified electrodes was next incubated with BSA (50 µL, 0.1 mM). Lastly, before being used, modified GCEs were left in air at 4°C. 2.5. Electrochemical characterization The modified GCE was submerged in different NSE solution concentrations and maintained for 20 minutes at 37°C in order to perform electrochemical detection. The nonspecific binding cells were then eliminated from the electrode by rinsing it with deionized water. A electrochemical workstation (CHI660E, CH Instrument Company, Shanghai, China) with a three-electrode system—a Pt foil as an auxiliary the electrode, Ag/AgCl(1M KCl) to be the reference-electrode, and the working electrode is either bare or modified GCE—was used to perform the electrochemical measures of differential pulse voltammetry(DPV), cyclic voltammetry(CV), and electrochemical impedance spectroscopy(EIS). Every electrochemical investigation was carried out with a 0.1 M PBS (1:1) having 5mM [Fe(CN) 6 ] 3−/4− solutions present. The scan rate for the CV and DPV studies was 50 mV/s. DPV measurements were performed with a 50mV amplitude, a 30 ms pulse width, and a 6mV step potential. At an AC amplitude of 5 mV, EIS measurements were conducted at frequencies between 10 − 1 and 105 Hz. 3. Results and discussions 3.1. Synthetic nanomaterial characterization Figures 1 A and 1 B show SEM and TEM images of PTCA functionalized CCG, respectively. On the surface of CCG nanosheets, dark nebulas (PTCA clusters) are uniformly absorbed through robust interactions with the hydroxyl and epoxy groups (He et al. 2018 ). In the PTCA/CCG, the single-layer and few-layer CCG nanosheets created a three-dimensional porous network that can increase its effective surface area. Figure 2 A shows the XRD patterns of GO, CCG, PTCA and PTCA/CCG. The diffraction peak is located at 2θ = 10.81° (002) and 26.44° (002) in the XRD patterns of GO and CCG. The separations between the diffraction peaks of GO and CCG show that the distance between the layers between the graphene sheets changes when hydrazine chemically reduces oxygen-containing groups and when the epoxides' ring-opening reaction produces aminoaziridine moieties that are bigger than the GO surfaces' epoxy groups (Geng et al. 2010 ). The XRD pattern of PTCA shows the diffraction peaks at 2θ = 22.71° (102) and 27.54°(201), corresponding of crystallites of the β-form of PTCA (Shulitski and Filippov 2009 ; Song et al. 2019 ). A weak crystalline structure of CCG predominant for PTCA-CCG is confirmed by the XRD pattern of PTCA/CCG, which displays a dominating weak-crystalline pattern of CCG. PTCA molecules are stacked face-to-face on CCG because to the π-π contact between the five-benzene nucleus of PTCA and the sp 2 plane of CCG (Gan et al. 2016 ). Figure 2 B displays the FT-IR spectrums of GO,CCG, PTCA and PTCA/CCG. FT-IR spectrum for GO exhibits a strong peak at 3425 cm − 1 ascribed to OH group, and a peak at 1560 cm − 1 that is ascribed to oxygen-aromatic binding within the aromatic ether or stretching of linked C–C within the aromatic ring (Nasir et al. 2017 ). The observed lower intensity peaks at 1735, 1620, 1395, 1215, and 1045 cm − 1 are assigned to the carboxylic acid groups (C = O) (Alam et al. 2017 ), the skeletal stretching of C = C alkene group (Sharma et al. 2017 ), O-H deformation vibration (Yaghoubidoust et al. 2014 ), C-O stretching (Zojaji et al. 2021 ), and stretching vibration of epoxy and alkoxy groups (Yadav et al. 2016 ), respectively. The CCG sample showed degradation in the groups of carboxylic acids as the peak on 3425 cm − 1 and was peak on 1735 cm − 1 lost strength (Faniyi et al. 2019 ). Additionally, the peak shows up at lower waves of CCG (1555cm − 1 ) than GO(1625cm − 1 ), indicating that the linked aromatic networks have been restored (An et al. 2017 ). Following reduction, the FT-IR data shows that the remaining oxygen-containing functional groups are epoxy and hydroxyl groups. The stretching vibration generated by the -OH and C = O groups, that are formed from the carboxyl groups of PTCA, can be linked to the peaks on 3435 and 1730 cm-1 in the FT-IR electromagnetic spectrum of PTCA (Ma et al. 2020 ). The aromatic ring's C═C stretching vibration is represented by the peak at 1660 cm –1 (Ma et al. 2020 ). Both the distinctive peaks for CCG and PTCA are visible in the FT-IR spectra of PTCA/CCG. In addition, FT-IR spectrum of PTCA/CCG displays slightly blue-shift compared to PTCA, indicating π-π stacking between CCG and PTCA and successful functionalization of the CCG by PTCA via π– π interactions between the pyrenyl group of PTCA and the surface of the CCG (Zhu et al. 2012 ). Figure 2 C present survey XPS spectra of GO, CCG, PTCA and PTCA/CCG. As seen, the survey XPS spectra of GO and PTCA show the peaks related to C 1s and O 1s, and the survey XPS spectra of CCG and PTCA/CCG show the additional peak related to N1s. For CCG and PTCA/CCG, it is found that the C/O ratio is considerably increased after reduction, because of the removal of oxygen-containing functional groups (Wan et al. 2013 ). The appearance of the N1s peak in CCG and PTCA/CCG samples can be associated with the nitrogen doping by hydrazine monohydrate during the reduction process (Chang et al. 2013 ). Figure 2 D displays the C1s XPS for GO, CCG, and PTCA/CCG. Two distinctive peaks in 284.5 and 286.4eV are explained by C-C and C-O species in the C 1s XPS of GO, whereas two faint peaks in 287.5 and 288.2eV are associated with C = O andO-C = O species (Shen et al. 2020 ). The peaks represent the existence of carboxylic, carbonyl, epoxy, and hydroxyl acid group on the GO sheets' edges and surfaces (Geng and Jung 2010 ). The peak energies at 288.2 and 287.5 eV dropped after chemical reduction in the CCG and PTCA/CCG examples, demonstrating that GO reduction was effective in eliminating the oxygen-containing groups with functions, particularly hydroxyl or epoxy groups. Additionally, a novel peak that was observed from bond creation in hydrazine decrease at 285.7eV corresponds to a C-N species (Ramesh et al. 2021 ). 3.2. Electrochemical Responses of the aptasensor To assessment the electrochemical performance of the designed aptasensor, electrochemical measurements including EIS, CV and DPV were conducted. The CV responses of the modified and bare GCEs were monitored in 0.1M PBS with 5mM [Fe(CN) 6 ] 3−/4− solution, as shown in Fig. 3 A. Compared to the bare GCE, the CCG modified electrode exhibits pronounced redox peaks, credited to high surface-area of CCG nanosheets and graphene nanosheets's high intrinsic conductivity which facilitates rapid electron transfer between the sensor surface and electrolyte (Bai et al. 2011 ). For PTCA functionalized CCG modified electrode (PTCA/CCG/GCE), it is observed that the peak currents are increased compared to CCG/GCE because PTCA modifies graphene, improving the conductivity and allows for a stronger electrical signal. The conjugated π-system of PTCA facilitates efficient electron transfer between the electrolyte and the graphene surface (Feng et al. 2011 ). The separation of the reduction and oxidation peak potentials (ΔEp) for CCG/GCE and PTCA/CCG/GCE are around 59 mV, revealing a promising reversible electrochemical redox process. As seen, after incubation in aptamer solution (Apt/PTCA/CCG/GCE), the peak current is decreased, indicating the successful immobilization of aptamers on surface of PTCA/CCG/GCE. PTCA has functional groups such as carboxylic acids, which can react with the aptamer DNA (Mohagheghpour et al. 2023 ). Moreover, EDC/NHS coupling can form a covalent bond between carboxylic-acid group of PTCA and the amino groups or hydroxyl groups on the aptamer DNA (Smith et al. 2020 ). Moreover, the aromatic rings in the nucleotide bases of the DNA can stack on the CCG surface via π-π interactions (Radhika and Shankar 2024 ). Moreover, the incubation in BSA solution also decrease the peak current (BSA/Apt/PTCA/CCG/GCE). Immobilization of aptamers and BSA on surface of PTCA/CCG/GCE likely caused steric hindrance, partially inhibiting electron transfer (Sopoušek et al. 2021 ). Upon incubating the electrochemical aptasensor with NSE (NSE/BSA/Apt/PTCA/CCG/GCE), the peak currents further decreased because of formation of DNA aptamer-NSE complexes, resulting in increased steric hindrance (Jarczewska et al. 2018 ). Our electrochemical sensors were able to quantify NSE since the degree of current drop was directly related to the level of NSE. As the NSE concentration increased, more aptamer-NSE complexes formed, leading to a further decrease in peak current. EIS was employed as an important electrochemical approach to investigate the condition of the aptasensor at each step of its fabrication. Figure 3 B shows EIS spectra of bare and modified GCEs were recorded into 0.1M PBS with 5mM [Fe(CN) 6 ] 3−/4− . The obtained Nyquist curves consist of two main components: a semicircle at high frequency regions that diameter of the semicircle represents the charge-transfer resistance (Rct), and a straight line at low frequency that expresses diffusion process. As observed, Rct value of bare GCE is approximately 3169 Ω. The modification of GCE surface by PTCA/CCG resulted in a notable reduction of the Rct level to 63 Ω because of increase in conductivity, and the expansion of the active surface area, as previously approved by CV experiments. Subsequent immobilization of aptamer and BSA onto the PTCA/CCG/GCE lead to a remarkable increase in the semicircle's diameter in the Nyquist plots and decrease of Rct of Apt/PTCA/CCG/GCE (2885 Ω) and BSA/Apt/PTCA/CCG/GCE (3645 Ω), revealing a blockage of charge transfer between the GCE and the redox probe. Finally, the interaction between NSE and the aptamer results a significant increase in the Rct to 3977 Ω, substantiating the successful interaction of the fabricated aptasensor with NSE. Figure 3 C illustrates the DPV responses of the bare and modified electrodes. The PTCA/CCG/GCE shows maximum peak current because of the efficient redox activity of PTCA and the excellent conductivity of CCG. However, because aptamers reduce the efficiency of electron transport across the redox probes and sensing interface, their insertion resulted in a drop of the redox peak current (Beiranvand and Azadbakht 2017 ). Upon incubation with a 0.1ng/mL NSE solution, the insulating layers on the electrode further inhibited electron transfer, decreasing the peak currents which is proportional to the concentration of NSE, thereby allowing the proposed aptasensor to detect NSE (Upan et al. 2021 ). 3.3. Optimization of Experimental Variables The amount of PTCA/CCG, incubation time of aptamer and analyte are important for both capturing lung cancer specific biomarker and enhancing the performance of the aptasensor. The effects of incubation time for aptamer and NSE were investigated for determination fixed concentration of NSE (0.1 ng/mL). As exhibited in Figs. 3 D and 3 E, the electrochemical signal gradually increased with longer incubation times and reach to maximum at 3 hours for incubation of aptamer and at 20 minutes for incubation of NSE. For longer time, it reached a steady state, presumably caused by the saturation of aptamers and analyte. The results indicate that 3 hours and 20 minutes are the optimum incubation time of aptamer and analyte, respectively, and these incubations time are sufficient for thorough capture of NSE onto modified-electrode surface. The amount of PTCA/CCG-nafion solution used to prepare PTCA/CCG modified GCE was also examined by tracking the DPV signal response with the intention of enhance the signal responsiveness. Figure 3 F shows that the peak current increased gradually with increasing amount of PTCA/CCG-nafion solution, reaching a maximum at 10 µL before leveling off between 10–40 µL. Thus, 10 µL was chosen as the optimal amount of PTCA/CCG-nafion solution for PTCA/CCG modification GCE. 3. 4. Quantitative Detection of NSE To study the linear range and sensitivity for the electrochemical aptasensor to detect NSE, DPV curve response of BSA/Apt/PTCA/CCG/GCE toward various concentrations of NSE (0 to 10 3 ng/mL) were tested. Figure 4 A exhibits that the DPV curve responses increased remarkably with the rise in NSE concentration from 0 to 10 3 ng/mL. A calibration curve between the f peak current shift and the logarithm for the target concentration, which ranges from 10 − 3 to 10 3 ng/mL, is generated. Figure 4 B shows that the peak current variation exhibits a logarithmic relationship to the concentration of NSE. A detection limit (LOD) that is as low as 0.008 ng/mL is based on the computation of 3σ/slope, where σ is the average deviation from five blank samples. As shown in Table 1 , the suggested electrochemical aptasensor performance for NSE detection is superior to that of other published biosensors. The findings demonstrate that a more sensitive substrate for identifying NSE is provided by the existing aptasensor. Table 1 Comparison between the performance of the proposed electrochemical aptasensor and reported biosensors for NSE detection. Analysis methods Material used linear range (ng/mL) LOD (ng/mL) Ref. LC–MS/MS -------- 5–500 0.038 (Torsetnes et al. 2013 ) LC-MS/MS Molecularly imprinted polymers 7.5–375 1.8 (McKitterick et al. 2020 ) SPFS Polydopamine coated plasmonic chip 1 − 100 0.5 (Toma et al. 2018 ) CLM Antibody/paramagnetic microparticles, and acridinium-labeled Antibody/Siemens ADVIA Centaur XPT system 1.6 − 400 1.6 (Cline et al. 2024 ) CLEI Fluorescein isothiocyanate labeled antibody 0–300 0.2 (Fu et al. 2012 ) ECLI Polyluminol and glucose oxidase-conjugated glucose-encapsulating liposome 10 − 4 −100 1.28×10 − 5 (Khakzad Aghdash et al. 2022 ) EC Amino functional graphene/thionine/Au NPs 1 − 500 0.01 (Fan et al. 2017 ) EC Au nanostructured electrode 1 − 750 0.34 (Sadrjavadi et al. 2022 ) EC hemin/reduced graphene oxide/multi-walled carbon nanotube 0.1 − 100 0.011 (Wei et al. 2024 ) EC 3D macroporous reduced graphene oxide/polyaniline film 0.005 − 10 10 − 3 (Zhang et al. 2018 ) EC Guanine-decorated graphene nanostructures 0.005 − 80 0.01 (Guang-Zhou Li and Tian 2013 ) EC BSA/Apt/PTCA/CCG/GCE 10 − 3 −10 3 0.008 This work 3.5. Reproducibility, stability, repeatability, and selectivity of the aptasensor When examined on four different fabricated aptasensors (BSA/Apt/PTCA/CCG/GCEs) under the same condition, the results of DPV measurement for determination NSE (0.1ng/mL) in Fig. 5 A illustrate an acceptable relative standard deviation (RSD) value of 3.38%, highlighting the reproducibility of the test under experimental conditions. Moreover, the stability and repeatability of the fabricated aptasensor was evaluated using BSA/Apt/PTCA/CCG/GCE stored at 4°C for 30 days. Figure 5 B shows that the DPV signal experienced less than a 3.43% decline compared with the initial response, indicating that no remarkable decomposition occurred, and aptasensor remained stable during long-term storage. Moreover, this indicates that proposed aptasensor possess promising repeatability. To assess the selectivity of the current aptasensor, the DPV responses to NSE (0.1ng/mL) were studied in the presence of 10 ng/mL carcinoembryonic antigen (CEA), immunoglobulin G (IgG), alpha fetoprotein (AFP), cytokeratin 19 fragment (CYFRA 21 − 1), alkaline phosphatase (ALP) and lysozyme as interfering species. The results in Fig. 5 C demonstrate that, even when there is a significant concentration of interfering species present, the aptasensor exhibits selective electrochemical responses to the NSE. The stability is attributed to the combination of CCG and PTCA which provides a robust platform for aptamer immobilization. CCG provides a large, conductive surface for the aptamer to adsorb onto, facilitating efficient electron transfer and high sensitivity, and the strong π-π interactions and hydrophobic effects ensure that the aptamer is stably adsorbed onto the CCG surface (Sekhon et al. 2021 ). Amine-terminated aptamers are covalently coupled to carboxylic-acid group of the PTCA/CCG composite onto the surface of electrode and subsequently hybridized with aptamer DNA (Hosseinzadeh and Mazloum-Ardakani 2020 ), which enhances selectivity and long-term stability, even under harsh environmental conditions. The adsorption does not disrupt the secondary structure of the aptamer, allowing it to retain its binding affinity for the target molecule. It is assumed that one aptamer binds to one specific antigen on the cell surface. This allows aptamers to distinguish cell type, malignant status, cancer type, proliferation capability, and metastatic potential. In addition, owing to their high specificity for targets, low toxicity and many advantages over other biological recognition elements, aptadensors can be considered appropriate for cancer cell recognition (Hosseinzadeh and Mazloum-Ardakani 2020 ). The use of EDC/NHS coupling ensures a strong and stable amide covalent bond between the aptamer DNA and carboxylic groups of PTCA (Hao Wu et al. 2020 ). Furthermore, incubation in BSA minimize undesirable surface interaction and nonspecific binding to cells (Yasun et al. 2015 ). 3.6. Examination of real serum specimens Human serum samples were spiked with various analyte contents (0.00, 0.50, 1.00, and 2.00 ng/mL) in order to assess the aptadensor's dependability and possible real-world uses. Prior to the NSE injection, the serum specimens were diluted thirty times. The compiled findings in Table 2 show that the fabricated aptadensor's precision and dependability are satisfactory, with recovery rates in blood specimens falling between 98.00% and 99.00% and an RSD of less than 4.08%. The reference ELISA method was used to measure the analyte concentrations in order to further assess the validity and accuracy of the suggested aptadensor when used in clinical serum specimens. A robust correlation between those two approaches supported the developed aptadensor's prospective clinical usage, as shown in Table 2 , indicating the newly developed BSA/Apt/PTCA/CCG/GCE aptadensor may find utility in clinical settings. Table 2 Results of the electrochemical aptasensor used to detect NSE in serum specimens (n = 3). Aptasensor ELISA Sample Added (ng/mL) Found (ng/mL) Recovery (%) RSD (%) Found (ng/mL) Recovery (%) RSD (%) Relative difference (%) Human serum 0.00 0.00 --- 3.72 0.00 --- 4.01 --- 0.50 0.49 98.00 3.97 0.49 98.00 3.22 0.0000 1.00 0.98 98.00 4.11 0.99 99.00 4.41 1.0101 2.00 1.98 99.00 4.08 1.97 98.50 4.19 -0.5076 4. Conclusions This study developed a new electrochemical aptasensor to identify NSE as a lung cancer biomarker in human serum samples in a selective, sensitive, and accurate manner. The aptasensor was relied on a NSE aptamer immobilized on PTCA/CCG modified electrode that it exhibited broad linear-range of 10 − 3 to 10 3 ng/mL with a significant low LOD of 0.008 ng/mL. The study's main findings demonstrated the improved sensitivity, selectivity and long-term stability of the electrochemical aptasensor, revealing its potential for early lung cancer diagnosis and monitoring. Future research could focus on optimizing the nanomaterial structure to further promote performance of electrochemical aptasensor, and developing low-cost and miniaturized point-of-care cancer diagnostics. The significant sensitive and selective aptasensor developed in this wok provides remarkable promise for early-stage lung cancer detection and monitoring, offering an accurate sensing platform for enhanced patient care and disease management. Declarations Conflict of interest All authors declare that they have no conflict of interest. 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Adv Colloid Interface Sci 289:102314 Cite Share Download PDF Status: Published Journal Publication published 29 Sep, 2025 Read the published version in Chemical Papers → Version 1 posted Reviewers agreed at journal 20 Apr, 2025 Reviewers invited by journal 22 Mar, 2025 Editor invited by journal 13 Mar, 2025 Editor assigned by journal 08 Mar, 2025 First submitted to journal 06 Mar, 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6166131","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":432453339,"identity":"916ef856-84da-4633-a270-5d3d83fc55a1","order_by":0,"name":"Jiqun Geng","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Jiqun","middleName":"","lastName":"Geng","suffix":""},{"id":432453340,"identity":"842d89af-cdf2-487a-8afe-c5c5724cf3e3","order_by":1,"name":"Xiansong Yang","email":"","orcid":"","institution":"Qingdao Medical College: Qingdao University Medical College","correspondingAuthor":false,"prefix":"","firstName":"Xiansong","middleName":"","lastName":"Yang","suffix":""},{"id":432453341,"identity":"e9957011-a78b-4b78-b8ae-90d7d268eb79","order_by":2,"name":"Ye Wang","email":"","orcid":"","institution":"Qingdao Medical College: Qingdao University Medical College","correspondingAuthor":false,"prefix":"","firstName":"Ye","middleName":"","lastName":"Wang","suffix":""},{"id":432453342,"identity":"f8134511-a88e-40d9-9e23-24da7c3ad7bb","order_by":3,"name":"Jun Che","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Che","suffix":""},{"id":432453343,"identity":"95ab7025-b2bf-4138-9094-2ac18ce2b9dc","order_by":4,"name":"Minwei Gu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsUlEQVRIiWNgGAWjYJCCA0DMw8/MfPgBaVok29nSDEizyuA8j4IEUSrlI7ITDxe22cgYH+ZhMGCosYkmqMXwRu6GwzPb0njMDvMeeMBwLC23gaCWGUAtvNsOA7XwJRgwNhwmWst/HuNmHgMJorTIS4C1HOAxYCZWiwHPW6CWf8k8EoeBgZxAjF/k23M3f+Y5Y2fP33/48IMPNTZE2HIAmZdASDnYFoKGjoJRMApGwSgAACLaPlIpZ/NPAAAAAElFTkSuQmCC","orcid":"","institution":"Affiliated Hospital of Jiangnan University","correspondingAuthor":true,"prefix":"","firstName":"Minwei","middleName":"","lastName":"Gu","suffix":""}],"badges":[],"createdAt":"2025-03-06 01:44:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6166131/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6166131/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11696-025-04382-0","type":"published","date":"2025-09-29T15:56:57+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79597599,"identity":"2f6cb0ba-13be-4655-b237-45ec79ad8119","added_by":"auto","created_at":"2025-03-31 14:27:07","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":250162,"visible":true,"origin":"","legend":"\u003cp\u003e(A) SEM and (B)TEM images of PTCA/CCG.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6166131/v1/158b77412d3cff1432b3e95c.jpeg"},{"id":79597602,"identity":"d073d941-08ec-41ea-93bd-ad9b59a3c0b4","added_by":"auto","created_at":"2025-03-31 14:27:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":513228,"visible":true,"origin":"","legend":"\u003cp\u003e(A) The XRD patterns of GO, CCG, PTCA and PTCA/CCG. (B) FT-IR spectrums of GO, CCG, PTCA and PTCA/CCG. (C) survey XPS spectra of GO, CCG, PTCA and PTCA/CCG, and (D) C 1s XPS of GO, CCG and PTCA/CCG.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6166131/v1/614fa1392043ee24c4c23aea.png"},{"id":79596653,"identity":"1099e7e9-4cbd-499a-b6e9-377594dd5ae9","added_by":"auto","created_at":"2025-03-31 14:19:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":722015,"visible":true,"origin":"","legend":"\u003cp\u003e(A) CV, (B) EIS and (C) DPV responses of the bare and modified GCEs. Optimization of the experimental parameters for Apt/PTCA/CCG/GCE: effects of (D) aptamer incubation time, (E) NSE incubation time, and (E) amount of PTCA/CCG-nafion solution on DPV signal response for determination of 0.1 ng/mL NSE into 0.1M PBS with 5mM [Fe(CN)\u003csub\u003e6\u003c/sub\u003e]\u003csup\u003e3−/4−\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6166131/v1/39d31f4c1ec3f5f2bc9e9633.png"},{"id":79596650,"identity":"61db29d6-19a9-451d-8963-9cb09aefe123","added_by":"auto","created_at":"2025-03-31 14:19:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":294029,"visible":true,"origin":"","legend":"\u003cp\u003e(A) The DPV curve responses of BSA/Apt/PTCA/CCG/GCE to NSE concentration from 0 to 10\u003csup\u003e3\u003c/sup\u003e ng/mL. (B) The logarithmic relationship between peak current and concentration of NSE.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6166131/v1/c29f453f7c8e7dbcec48bbad.png"},{"id":79596656,"identity":"9d5751b6-0192-4eaf-aa1a-34b6da31733a","added_by":"auto","created_at":"2025-03-31 14:19:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":111047,"visible":true,"origin":"","legend":"\u003cp\u003eThe DPV responses to NSE (0.1 ng/mL) for study (A) reproducibility, (B) stability, and (C) selectivity of the aptasensor in the presence of 10 ng/mL interfering species.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6166131/v1/c6d7ed9193de19452bf0e050.png"},{"id":92883735,"identity":"46799edb-31a1-4a92-9c9c-8d2859a82e8f","added_by":"auto","created_at":"2025-10-06 16:08:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2669679,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6166131/v1/a8b0ebe2-e83f-4808-8652-6d4e48c746ed.pdf"}],"financialInterests":"","formattedTitle":"Fabrication of Aptasensor for Monitoring cancer biomarkers: Detection of Lung Specific Biomarkers Using Chemical Converted Graphene","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eWhen cells in a particular area of the body multiply and reproduce excessively, it can lead to cancer. The malignant cells have the ability to spread and obliterate nearby healthy tissue, including organs (Alshammary and Al-Attar \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Therefore, the cancer is one of the most important health challenges worldwide, emphasizing the critical need for advancements in treatment, medication, and early diagnosis. By concentrating on identifying people with symptoms as soon as possible, early cancer detection typically improves the likelihood of a successful course of therapy. Recognition and description of cancer cells are greatly aided by a variety of cancer diagnostic techniques, such as laboratory testing, diagnostic imaging, endoscopic examinations, genetic testing, and tumor biopsies (Kohaar et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). To more reliably and consistently identify biomarkers for patient treatment, prognosis, medical facilities, and societal well-being, there is a rising need for the creation of more effective techniques that make use of bioinformatics, the internet, and molecular biology. By reducing needless procedures and allocating healthcare resources as efficiently as possible, biomarkers can improve the precision of cancer detection and treatment and increase economic efficiency (Ahmad et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA cancer biomarker might be either a biological molecule secreted by the tumor itself in bodily fluids such as blood or tissues which are the specific signs of a normal or abnormal process, or of a condition or disease such as cancer (Zakari et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Specifically, lung cancer, one of the leading causes of cancer-related deaths, has its own set of biomarkers that these included pro-gastrin-releasing peptides(ProGRP), Squamous cells carcinoma-associated antigen(SCC), carcinoembryonic antigen (CEA), cytokeratin-19 fragment(CYFRA), and sialyl Lewis-X antigen(SLX) and neuron-specific enolase (NSE) (Mizuno et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Between them, studied have been indicated that NSE is as an important biomarker for lung cancer, demonstrating the vital of its detection and patients having suspected lung cancer frequently have it measured (Huang et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAptamers are now biocompatible tools that can be utilized to find protein biomarkers. Short, single-stranded oligonucleotides called aptamers (DNA or RNA) fold into tertiary structures to attach to targets with great affinity and selectivity (Lingling Wu et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Aptamers are used as identification elements in aptamer-based biosensors, or aptasensors, to detect a target, which is frequently connected to a readable signal. Aptamers-based probes are essential for precisely targeting cancer biomarkers (Kulabhusan et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Aptamer-cancer biomarker affinity becomes crucial for targeted therapy, prognosis tracking, and diagnosis.\u003c/p\u003e \u003cp\u003eAptasensors' improved stability, adaptability, and capacity for regeneration for reusable biosensors are their primary advantages over immunosensors (Arshavsky-Graham et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). According to studies, aptasensors have a noticeably longer shelf life than immunosensors and may be kept in dry settings and for a while without losing their sensitivity (Al Mamun et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Arshavsky-Graham et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Arshavsky‐Graham et al. 2022). Electrochemical aptasensors are unique among the several aptasensor kinds due to their quick reaction, affordability, and simplicity of downsizing (Radi \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBy utilizing the high signal amplification, distinct characteristics of different nanomaterials, and highly selective aptamer-target interactions, nanomaterial-based aptasensors are able to detect a variety of analytes of interest (Kurup et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Due to their enormous surface area and remarkable conductivity, nanostructures like graphene oxide (GO) can sustain substantial biomolecule loading for excellent detection sensitivity (Amiri et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Functionalized chemically converted graphene (CCG) offers additional benefits over traditional graphene oxide such as functionalization capabilities and improved stability (Bai et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Bottari et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe functionalized graphene sheets can be further altered by various chemical reactions, such as amidation, surface-initiated polymerization, which and reduction of metal ions, and they show better chemical and thermal stabilities than GO (Ismail et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition to being electrically conductive, the CCG nanosheets exhibit superior solvent dispersibility and processability (Kuila et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The incorporation of 3,4,9,10-perylene-tetracarboxylic-acid(PTCA) into CCG further improves the electrochemical properties of the aptasensor by providing better signal amplification and facilitating the electron transfer rate (Feng et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The synergetic effect of PTCA and CCG (PTCA/CCG) leads to a highly effective sensing platform for monitoring cancer biomarkers.\u003c/p\u003e \u003cp\u003eThe creation of a innovative electrochemical-aptasensor based on a modified PTCA/CCG electrode for the monitoring of lung-specific biomarkers for NSE was the main goal of this work. The selection of these materials is based on their exceptional properties and the potential to promote aptasensor performance. This research aims to provide a novel and sensitive platform for the early diagnosis of lung cancer, contributing to low cost, better diagnostic tools and enhanced patient outcomes.\u003c/p\u003e"},{"header":"2. Experiments","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eWe bought electrodes made of glassy carbon (GCE, 5mm diameter) from Tokai Electrode Mfg. Co., Japan Alumina powder (1.0, 0.3, and 0.05 \u0026micro;m) was obtained from Buehler, USA. Ultrapage purified aptamer for NSE (5\u0026rsquo;- biotin-TEG-TCACACACGGACCTCTC CTACATTAATTGCGCATTTCGTT-3\u0026rsquo;) was ordered from Sangon Biotechnology Co. Recombinant human carcinoembryonic antigen (CEA) was purchased from Sangon Biotech, China. Alphafetoprotein (AFP) antigen and recombinant neuron specific enolase (NSE) protein were provided from Shanghai LincBio Science Co. Ltd., China. 1-ethyl-3- (3-dimethylaminopropyl)-carbodimide hydrochloride (EDC) was sourced from Pierce Biotechnology, Inc. (Rockford, USA). Alkaline phosphatase (ALP) was obtained from BBI Enzymes (Wisconsin, USA). N-Hydroxysuccinimide(NHS), bovine serum albumin(BSA), ammonia aqueous solution (25 wt%), hydrazine solution, sodium hydroxide (NaOH), MES buffer, Sigma-Aldrich (St. Louis, MO, USA) provided the tris buffer, potassium chloride (KCl), magnesium chloride (MgCl2), phosphate-buffered saline (PBS), ferricyanide of potassium ([Fe(CN)6]\u003csup\u003e3\u0026minus;\u003c/sup\u003e), and potassium-ferrocyanide([Fe(CN)6]\u003csup\u003e4\u0026minus;\u003c/sup\u003e). Ethanol and acetone came from Merck in Germany. 3,4,9,10-Thermo Scientific Chemicals in the United States provided the ethylene tetracarboxylic dianhydride (PTCDA). A Milli-Q purification equipment was used in the lab to create deionized water.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Instrumentation\u003c/h2\u003e \u003cp\u003eDialysis membrane (Spectrapro membrane tubing, Spectrum Labs, CA, USA; MWCO 6\u0026ndash;8 kDa). An EQUINOX-55 FTIR spectrometer was used to acquire FT-IR spectra. To characterize the synthesized nanomaterial, X-ray diffraction (XRD, X'Pert PRO, PANalytical), transmission electron microscopy (TEM, Tecnai G2 F20, FEI), scanning electron microscopy (SEM, JSM-7100F, JEOL Ltd., Japan), and X-ray photoelectron spectra (XPS) were employed. The actual samples were analyzed using the Human Neuron specific Enolase enzyme-linked immunosorbent test (ELISA) Kit (ab217778, Abcam).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Preparation of CCG and PTCA/CCG\u003c/h2\u003e \u003cp\u003eCCG was prepared according to the procedure reported by Li et al. (Dan Li et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Briefly, ammonia aquatic solution (80 \u0026micro;L, 25 wt%) and hydrazine solution (5 \u0026micro;L) were added into the suspension of the GO (5 mL, 1 mg/mL). After 8 minutes magnetic stirring, the vial was placed in a water bath (90\u0026deg;C) for 60 minutes under vigorous stirring. For preparation of the PTCA/CCG, this procedure was conducted using mixture of ammonia aquatic solution, hydrazine solution, suspension of the GO and PTCA. PTCA was formed by hydrolyzing PTCAD (0.3 M) in sodium hydroxide (100 mL, 1M). a mixture of 3.92 g of PTCAD and NaOH was refluxed for 4 hours to obtain yellow-green solution (Sun et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Following centrifugation, the solid was recovered and vacuum-dried at 37\u0026deg;C. A mixture of GO suspension (5 mL, 1 mg/mL) and PTCA powder (20 mg) was prepared by adding water to a final volume of 100 mL. After 30 minutes ultrasonication, the mixture was vigorously stirred at 40\u0026deg;C overnight. After that, hydrazine solution (5 \u0026micro;L) and ammonia solution (80 \u0026micro;L, 25 wt%) were added to the mixture. The resulted mixture was then heated at 90\u0026deg;C for 60 minutes under magnetic stirring. Next, the product was dialyzed using dialysis membrane for 3 days; dialysis water was changed 3 times daily. Finally, the precipitate was removed to reach the stable homogeneous solution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Modification of Electrodes with Aptamer, CCG, and PTCA/CCG\u003c/h2\u003e \u003cp\u003eBefore modification GCEs, GCEs were polished sequentially with 1.0, 0.3, and 0.05 \u0026micro;m Alumina powder, 5 minutes for each powder. Then, GCEs were sonicated for 10 minutes each in water, acetone and ethanol, and then dried under a directed stream of high-purity nitrogen. For modification, the PTCA/CCG mixture was mixed by a nafion solution (0.1%) in a 4:1 volume ratio to improve the affinity of the composite with the GCE. Then, PTCA/CCG-nafion solution (10 \u0026micro;L) was drop-casted onto the GCE and dried at 37\u0026deg;C. The carboxyl groups were then activated for 60 minutes by applying an activation solution that contained NHS (0.2 M) and EDC (0.1 M) into MES buffer (0.1 M). After that, the electrode surface was rinsed with 10 mM Tris buffer (pH\u0026thinsp;=\u0026thinsp;7.4). The electrode surface was then drop-cast with aptamer (10 \u0026micro;L, 0.1 mM) containing a terminal amine group, and it was incubated for three hours. Afterwards, the modified electrode surface were washed with buffer and then hybridized by 1 mM of aptamer in solution (pH\u0026thinsp;=\u0026thinsp;7.4) containing Tris buffer (10 mM), MgCl\u003csub\u003e2\u003c/sub\u003e (2.5 mM), KCl (140 mM) for 60 minutes. To block the remainder of bonding sites on the PTCA/CCG surface, the modified electrodes was next incubated with BSA (50 \u0026micro;L, 0.1 mM). Lastly, before being used, modified GCEs were left in air at 4\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Electrochemical characterization\u003c/h2\u003e \u003cp\u003eThe modified GCE was submerged in different NSE solution concentrations and maintained for 20 minutes at 37\u0026deg;C in order to perform electrochemical detection. The nonspecific binding cells were then eliminated from the electrode by rinsing it with deionized water. A electrochemical workstation (CHI660E, CH Instrument Company, Shanghai, China) with a three-electrode system\u0026mdash;a Pt foil as an auxiliary the electrode, Ag/AgCl(1M KCl) to be the reference-electrode, and the working electrode is either bare or modified GCE\u0026mdash;was used to perform the electrochemical measures of differential pulse voltammetry(DPV), cyclic voltammetry(CV), and electrochemical impedance spectroscopy(EIS). Every electrochemical investigation was carried out with a 0.1 M PBS (1:1) having 5mM [Fe(CN)\u003csub\u003e6\u003c/sub\u003e]\u003csup\u003e3\u0026minus;/4\u0026minus;\u003c/sup\u003e solutions present. The scan rate for the CV and DPV studies was 50 mV/s. DPV measurements were performed with a 50mV amplitude, a 30 ms pulse width, and a 6mV step potential. At an AC amplitude of 5 mV, EIS measurements were conducted at frequencies between 10\u0026thinsp;\u0026minus;\u0026thinsp;1 and 105 Hz.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussions","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Synthetic nanomaterial characterization\u003c/h2\u003e \u003cp\u003eFigures \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB show SEM and TEM images of PTCA functionalized CCG, respectively. On the surface of CCG nanosheets, dark nebulas (PTCA clusters) are uniformly absorbed through robust interactions with the hydroxyl and epoxy groups (He et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In the PTCA/CCG, the single-layer and few-layer CCG nanosheets created a three-dimensional porous network that can increase its effective surface area.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA shows the XRD patterns of GO, CCG, PTCA and PTCA/CCG. The diffraction peak is located at 2θ\u0026thinsp;=\u0026thinsp;10.81\u0026deg; (002) and 26.44\u0026deg; (002) in the XRD patterns of GO and CCG. The separations between the diffraction peaks of GO and CCG show that the distance between the layers between the graphene sheets changes when hydrazine chemically reduces oxygen-containing groups and when the epoxides' ring-opening reaction produces aminoaziridine moieties that are bigger than the GO surfaces' epoxy groups (Geng et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The XRD pattern of PTCA shows the diffraction peaks at 2θ\u0026thinsp;=\u0026thinsp;22.71\u0026deg; (102) and 27.54\u0026deg;(201), corresponding of crystallites of the β-form of PTCA (Shulitski and Filippov \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Song et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). A weak crystalline structure of CCG predominant for PTCA-CCG is confirmed by the XRD pattern of PTCA/CCG, which displays a dominating weak-crystalline pattern of CCG. PTCA molecules are stacked face-to-face on CCG because to the π-π contact between the five-benzene nucleus of PTCA and the sp\u003csup\u003e2\u003c/sup\u003e plane of CCG (Gan et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB displays the FT-IR spectrums of GO,CCG, PTCA and PTCA/CCG. FT-IR spectrum for GO exhibits a strong peak at 3425 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ascribed to OH group, and a peak at 1560 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e that is ascribed to oxygen-aromatic binding within the aromatic ether or stretching of linked C\u0026ndash;C within the aromatic ring (Nasir et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The observed lower intensity peaks at 1735, 1620, 1395, 1215, and 1045 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are assigned to the carboxylic acid groups (C\u0026thinsp;=\u0026thinsp;O) (Alam et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), the skeletal stretching of C\u0026thinsp;=\u0026thinsp;C alkene group (Sharma et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), O-H deformation vibration (Yaghoubidoust et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), C-O stretching (Zojaji et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and stretching vibration of epoxy and alkoxy groups (Yadav et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), respectively. The CCG sample showed degradation in the groups of carboxylic acids as the peak on 3425 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and was peak on 1735 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e lost strength (Faniyi et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Additionally, the peak shows up at lower waves of CCG (1555cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) than GO(1625cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), indicating that the linked aromatic networks have been restored (An et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Following reduction, the FT-IR data shows that the remaining oxygen-containing functional groups are epoxy and hydroxyl groups. The stretching vibration generated by the -OH and C\u0026thinsp;=\u0026thinsp;O groups, that are formed from the carboxyl groups of PTCA, can be linked to the peaks on 3435 and 1730 cm-1 in the FT-IR electromagnetic spectrum of PTCA (Ma et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The aromatic ring's C═C stretching vibration is represented by the peak at 1660 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e (Ma et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Both the distinctive peaks for CCG and PTCA are visible in the FT-IR spectra of PTCA/CCG. In addition, FT-IR spectrum of PTCA/CCG displays slightly blue-shift compared to PTCA, indicating π-π stacking between CCG and PTCA and successful functionalization of the CCG by PTCA via π\u0026ndash; π interactions between the pyrenyl group of PTCA and the surface of the CCG (Zhu et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC present survey XPS spectra of GO, CCG, PTCA and PTCA/CCG. As seen, the survey XPS spectra of GO and PTCA show the peaks related to C 1s and O 1s, and the survey XPS spectra of CCG and PTCA/CCG show the additional peak related to N1s. For CCG and PTCA/CCG, it is found that the C/O ratio is considerably increased after reduction, because of the removal of oxygen-containing functional groups (Wan et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The appearance of the N1s peak in CCG and PTCA/CCG samples can be associated with the nitrogen doping by hydrazine monohydrate during the reduction process (Chang et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD displays the C1s XPS for GO, CCG, and PTCA/CCG. Two distinctive peaks in 284.5 and 286.4eV are explained by C-C and C-O species in the C 1s XPS of GO, whereas two faint peaks in 287.5 and 288.2eV are associated with C\u0026thinsp;=\u0026thinsp;O andO-C\u0026thinsp;=\u0026thinsp;O species (Shen et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The peaks represent the existence of carboxylic, carbonyl, epoxy, and hydroxyl acid group on the GO sheets' edges and surfaces (Geng and Jung \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The peak energies at 288.2 and 287.5 eV dropped after chemical reduction in the CCG and PTCA/CCG examples, demonstrating that GO reduction was effective in eliminating the oxygen-containing groups with functions, particularly hydroxyl or epoxy groups. Additionally, a novel peak that was observed from bond creation in hydrazine decrease at 285.7eV corresponds to a C-N species (Ramesh et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Electrochemical Responses of the aptasensor\u003c/h2\u003e \u003cp\u003eTo assessment the electrochemical performance of the designed aptasensor, electrochemical measurements including EIS, CV and DPV were conducted. The CV responses of the modified and bare GCEs were monitored in 0.1M PBS with 5mM [Fe(CN)\u003csub\u003e6\u003c/sub\u003e]\u003csup\u003e3\u0026minus;/4\u0026minus;\u003c/sup\u003e solution, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. Compared to the bare GCE, the CCG modified electrode exhibits pronounced redox peaks, credited to high surface-area of CCG nanosheets and graphene nanosheets's high intrinsic conductivity which facilitates rapid electron transfer between the sensor surface and electrolyte (Bai et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). For PTCA functionalized CCG modified electrode (PTCA/CCG/GCE), it is observed that the peak currents are increased compared to CCG/GCE because PTCA modifies graphene, improving the conductivity and allows for a stronger electrical signal. The conjugated π-system of PTCA facilitates efficient electron transfer between the electrolyte and the graphene surface (Feng et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The separation of the reduction and oxidation peak potentials (ΔEp) for CCG/GCE and PTCA/CCG/GCE are around 59 mV, revealing a promising reversible electrochemical redox process. As seen, after incubation in aptamer solution (Apt/PTCA/CCG/GCE), the peak current is decreased, indicating the successful immobilization of aptamers on surface of PTCA/CCG/GCE. PTCA has functional groups such as carboxylic acids, which can react with the aptamer DNA (Mohagheghpour et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Moreover, EDC/NHS coupling can form a covalent bond between carboxylic-acid group of PTCA and the amino groups or hydroxyl groups on the aptamer DNA (Smith et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Moreover, the aromatic rings in the nucleotide bases of the DNA can stack on the CCG surface via π-π interactions (Radhika and Shankar \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Moreover, the incubation in BSA solution also decrease the peak current (BSA/Apt/PTCA/CCG/GCE). Immobilization of aptamers and BSA on surface of PTCA/CCG/GCE likely caused steric hindrance, partially inhibiting electron transfer (Sopoušek et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Upon incubating the electrochemical aptasensor with NSE (NSE/BSA/Apt/PTCA/CCG/GCE), the peak currents further decreased because of formation of DNA aptamer-NSE complexes, resulting in increased steric hindrance (Jarczewska et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Our electrochemical sensors were able to quantify NSE since the degree of current drop was directly related to the level of NSE. As the NSE concentration increased, more aptamer-NSE complexes formed, leading to a further decrease in peak current.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEIS was employed as an important electrochemical approach to investigate the condition of the aptasensor at each step of its fabrication. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB shows EIS spectra of bare and modified GCEs were recorded into 0.1M PBS with 5mM [Fe(CN)\u003csub\u003e6\u003c/sub\u003e]\u003csup\u003e3\u0026minus;/4\u0026minus;\u003c/sup\u003e. The obtained Nyquist curves consist of two main components: a semicircle at high frequency regions that diameter of the semicircle represents the charge-transfer resistance (Rct), and a straight line at low frequency that expresses diffusion process. As observed, Rct value of bare GCE is approximately 3169 Ω. The modification of GCE surface by PTCA/CCG resulted in a notable reduction of the Rct level to 63 Ω because of increase in conductivity, and the expansion of the active surface area, as previously approved by CV experiments. Subsequent immobilization of aptamer and BSA onto the PTCA/CCG/GCE lead to a remarkable increase in the semicircle's diameter in the Nyquist plots and decrease of Rct of Apt/PTCA/CCG/GCE (2885 Ω) and BSA/Apt/PTCA/CCG/GCE (3645 Ω), revealing a blockage of charge transfer between the GCE and the redox probe. Finally, the interaction between NSE and the aptamer results a significant increase in the Rct to 3977 Ω, substantiating the successful interaction of the fabricated aptasensor with NSE.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC illustrates the DPV responses of the bare and modified electrodes. The PTCA/CCG/GCE shows maximum peak current because of the efficient redox activity of PTCA and the excellent conductivity of CCG. However, because aptamers reduce the efficiency of electron transport across the redox probes and sensing interface, their insertion resulted in a drop of the redox peak current (Beiranvand and Azadbakht \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Upon incubation with a 0.1ng/mL NSE solution, the insulating layers on the electrode further inhibited electron transfer, decreasing the peak currents which is proportional to the concentration of NSE, thereby allowing the proposed aptasensor to detect NSE (Upan et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Optimization of Experimental Variables\u003c/h2\u003e \u003cp\u003eThe amount of PTCA/CCG, incubation time of aptamer and analyte are important for both capturing lung cancer specific biomarker and enhancing the performance of the aptasensor. The effects of incubation time for aptamer and NSE were investigated for determination fixed concentration of NSE (0.1 ng/mL). As exhibited in Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, the electrochemical signal gradually increased with longer incubation times and reach to maximum at 3 hours for incubation of aptamer and at 20 minutes for incubation of NSE. For longer time, it reached a steady state, presumably caused by the saturation of aptamers and analyte. The results indicate that 3 hours and 20 minutes are the optimum incubation time of aptamer and analyte, respectively, and these incubations time are sufficient for thorough capture of NSE onto modified-electrode surface.\u003c/p\u003e \u003cp\u003eThe amount of PTCA/CCG-nafion solution used to prepare PTCA/CCG modified GCE was also examined by tracking the DPV signal response with the intention of enhance the signal responsiveness. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF shows that the peak current increased gradually with increasing amount of PTCA/CCG-nafion solution, reaching a maximum at 10 \u0026micro;L before leveling off between 10\u0026ndash;40 \u0026micro;L. Thus, 10 \u0026micro;L was chosen as the optimal amount of PTCA/CCG-nafion solution for PTCA/CCG modification GCE.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e3. 4. Quantitative Detection of NSE\u003c/h3\u003e\n\u003cp\u003e \u003c/p\u003e \u003cp\u003eTo study the linear range and sensitivity for the electrochemical aptasensor to detect NSE, DPV curve response of BSA/Apt/PTCA/CCG/GCE toward various concentrations of NSE (0 to 10\u003csup\u003e3\u003c/sup\u003e ng/mL) were tested. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA exhibits that the DPV curve responses increased remarkably with the rise in NSE concentration from 0 to 10\u003csup\u003e3\u003c/sup\u003e ng/mL. A calibration curve between the f peak current shift and the logarithm for the target concentration, which ranges from 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e to 10\u003csup\u003e3\u003c/sup\u003e ng/mL, is generated. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB shows that the peak current variation exhibits a logarithmic relationship to the concentration of NSE. A detection limit (LOD) that is as low as 0.008 ng/mL is based on the computation of 3σ/slope, where σ is the average deviation from five blank samples. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the suggested electrochemical aptasensor performance for NSE detection is superior to that of other published biosensors. The findings demonstrate that a more sensitive substrate for identifying NSE is provided by the existing aptasensor.\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\u003eComparison between the performance of the proposed electrochemical aptasensor and reported biosensors for NSE detection.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnalysis methods\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaterial used\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003elinear range (ng/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLOD (ng/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRef.\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLC\u0026ndash;MS/MS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e--------\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026ndash;500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.038\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Torsetnes et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2013\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLC-MS/MS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMolecularly imprinted polymers\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.5\u0026ndash;375\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(McKitterick et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSPFS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePolydopamine coated plasmonic chip\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Toma et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2018\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCLM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAntibody/paramagnetic microparticles, and acridinium-labeled Antibody/Siemens ADVIA Centaur XPT system\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026minus;\u0026thinsp;400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Cline et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCLEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFluorescein isothiocyanate labeled antibody\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u0026ndash;300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Fu et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eECLI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePolyluminol and glucose oxidase-conjugated glucose-encapsulating liposome\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e\u0026minus;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.28\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Khakzad Aghdash et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAmino functional graphene/thionine/Au NPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Fan et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAu nanostructured electrode\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;750\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Sadrjavadi et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehemin/reduced graphene oxide/multi-walled carbon nanotube\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026minus;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Wei et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3D macroporous reduced graphene oxide/polyaniline film\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.005\u0026thinsp;\u0026minus;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Zhang et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2018\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGuanine-decorated graphene nanostructures\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.005\u0026thinsp;\u0026minus;\u0026thinsp;80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Guang-Zhou Li and Tian \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBSA/Apt/PTCA/CCG/GCE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e \u0026minus;10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis work\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Reproducibility, stability, repeatability, and selectivity of the aptasensor\u003c/h2\u003e \u003cp\u003eWhen examined on four different fabricated aptasensors (BSA/Apt/PTCA/CCG/GCEs) under the same condition, the results of DPV measurement for determination NSE (0.1ng/mL) in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA illustrate an acceptable relative standard deviation (RSD) value of 3.38%, highlighting the reproducibility of the test under experimental conditions. Moreover, the stability and repeatability of the fabricated aptasensor was evaluated using BSA/Apt/PTCA/CCG/GCE stored at 4\u0026deg;C for 30 days. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB shows that the DPV signal experienced less than a 3.43% decline compared with the initial response, indicating that no remarkable decomposition occurred, and aptasensor remained stable during long-term storage. Moreover, this indicates that proposed aptasensor possess promising repeatability.\u003c/p\u003e \u003cp\u003eTo assess the selectivity of the current aptasensor, the DPV responses to NSE (0.1ng/mL) were studied in the presence of 10 ng/mL carcinoembryonic antigen (CEA), immunoglobulin G (IgG), alpha fetoprotein (AFP), cytokeratin 19 fragment (CYFRA 21\u0026thinsp;\u0026minus;\u0026thinsp;1), alkaline phosphatase (ALP) and lysozyme as interfering species. The results in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC demonstrate that, even when there is a significant concentration of interfering species present, the aptasensor exhibits selective electrochemical responses to the NSE.\u003c/p\u003e \u003cp\u003eThe stability is attributed to the combination of CCG and PTCA which provides a robust platform for aptamer immobilization. CCG provides a large, conductive surface for the aptamer to adsorb onto, facilitating efficient electron transfer and high sensitivity, and the strong π-π interactions and hydrophobic effects ensure that the aptamer is stably adsorbed onto the CCG surface (Sekhon et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Amine-terminated aptamers are covalently coupled to carboxylic-acid group of the PTCA/CCG composite onto the surface of electrode and subsequently hybridized with aptamer DNA (Hosseinzadeh and Mazloum-Ardakani \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), which enhances selectivity and long-term stability, even under harsh environmental conditions. The adsorption does not disrupt the secondary structure of the aptamer, allowing it to retain its binding affinity for the target molecule. It is assumed that one aptamer binds to one specific antigen on the cell surface. This allows aptamers to distinguish cell type, malignant status, cancer type, proliferation capability, and metastatic potential. In addition, owing to their high specificity for targets, low toxicity and many advantages over other biological recognition elements, aptadensors can be considered appropriate for cancer cell recognition (Hosseinzadeh and Mazloum-Ardakani \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The use of EDC/NHS coupling ensures a strong and stable amide covalent bond between the aptamer DNA and carboxylic groups of PTCA (Hao Wu et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Furthermore, incubation in BSA minimize undesirable surface interaction and nonspecific binding to cells (Yasun et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Examination of real serum specimens\u003c/h2\u003e \u003cp\u003eHuman serum samples were spiked with various analyte contents (0.00, 0.50, 1.00, and 2.00 ng/mL) in order to assess the aptadensor's dependability and possible real-world uses. Prior to the NSE injection, the serum specimens were diluted thirty times. The compiled findings in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e show that the fabricated aptadensor's precision and dependability are satisfactory, with recovery rates in blood specimens falling between 98.00% and 99.00% and an RSD of less than 4.08%. The reference ELISA method was used to measure the analyte concentrations in order to further assess the validity and accuracy of the suggested aptadensor when used in clinical serum specimens. A robust correlation between those two approaches supported the developed aptadensor's prospective clinical usage, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, indicating the newly developed BSA/Apt/PTCA/CCG/GCE aptadensor may find utility in clinical settings.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults of the electrochemical aptasensor used to detect NSE in serum specimens (n\u0026thinsp;=\u0026thinsp;3).\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=\"char\" char=\".\" 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=\"left\" 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=\"left\" 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=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eAptasensor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c7\" namest=\"c4\"\u003e \u003cp\u003eELISA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAdded\u003c/p\u003e \u003cp\u003e(ng/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFound (ng/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRecovery (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRSD (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFound (ng/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRecovery (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eRSD (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eRelative difference (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eHuman serum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e98.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e98.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e98.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e99.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.0101\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e98.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-0.5076\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. Conclusions","content":"\u003cp\u003eThis study developed a new electrochemical aptasensor to identify NSE as a lung cancer biomarker in human serum samples in a selective, sensitive, and accurate manner. The aptasensor was relied on a NSE aptamer immobilized on PTCA/CCG modified electrode that it exhibited broad linear-range of 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e to 10\u003csup\u003e3\u003c/sup\u003e ng/mL with a significant low LOD of 0.008 ng/mL. The study's main findings demonstrated the improved sensitivity, selectivity and long-term stability of the electrochemical aptasensor, revealing its potential for early lung cancer diagnosis and monitoring. Future research could focus on optimizing the nanomaterial structure to further promote performance of electrochemical aptasensor, and developing low-cost and miniaturized point-of-care cancer diagnostics. The significant sensitive and selective aptasensor developed in this wok provides remarkable promise for early-stage lung cancer detection and monitoring, offering an accurate sensing platform for enhanced patient care and disease management.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eAll authors declare that they have no conflict of interest.\u003c/p\u003e \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAhmad I, Jasim SA, Sharma M, Hjazi A, Mohammed JS, Sinha A, Zwamel AH, Hamzah HF, Mohammed BA (2024) New paradigms to break barriers in early cancer detection for improved prognosis and treatment outcomes. 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Adv Colloid Interface Sci 289:102314\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":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"chemical-papers","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"chpa","sideBox":"Learn more about [Chemical Papers](http://link.springer.com/journal/11696)","snPcode":"11696","submissionUrl":"https://www.editorialmanager.com/CHPA/default.aspx","title":"Chemical Papers","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Lung Cancer Biomarker, Neuron Specific Enolase, Electrochemical Aptasensor, Chemical Converted Graphene, Stability","lastPublishedDoi":"10.21203/rs.3.rs-6166131/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6166131/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis work was focused on development a stable, selective and sensitive electrochemical aptasensor based on 3,4,9,10-perylene tetracarboxylic acid -functionalized chemical converted graphene (PTCA/CCG) modified electrode for monitoring neuron specific enolase (NSE), lung-specific biomarkers. The electrochemical aptasensor was relied on the purified NSE aptamer immobilized on PTCA/CCG modified electrode. The PTCA/CCG promoted both the immobilization of the aptamer and the electrochemical signal. The aptasensor exhibited a low limit of detection (0.008 ng/mL), broad linear range (10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e to 10\u003csup\u003e3\u003c/sup\u003e ng/mL), remarkable specificity, and excellent repeatability and long-term stability, maintaining initial resistance at 96.57% after 30 days. Results exhibited that the recoveries ranging from 98.00\u0026ndash;99.00% and relative standard deviation less than 4.08%, demonstrating the acceptable precision and reliability of the fabricated aptadensor and confirming the aptasensor's performance and efficiency in complex biological fluids and clinical applications.\u003c/p\u003e","manuscriptTitle":"Fabrication of Aptasensor for Monitoring cancer biomarkers: Detection of Lung Specific Biomarkers Using Chemical Converted Graphene","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-31 14:19:02","doi":"10.21203/rs.3.rs-6166131/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-04-20T09:58:56+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-22T11:59:25+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Chemical Papers","date":"2025-03-13T23:20:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-08T14:23:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Chemical Papers","date":"2025-03-06T16:18:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"chemical-papers","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"chpa","sideBox":"Learn more about [Chemical Papers](http://link.springer.com/journal/11696)","snPcode":"11696","submissionUrl":"https://www.editorialmanager.com/CHPA/default.aspx","title":"Chemical Papers","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"68938c47-ac27-45ef-9492-dff2bd53590e","owner":[],"postedDate":"March 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-06T16:01:10+00:00","versionOfRecord":{"articleIdentity":"rs-6166131","link":"https://doi.org/10.1007/s11696-025-04382-0","journal":{"identity":"chemical-papers","isVorOnly":false,"title":"Chemical Papers"},"publishedOn":"2025-09-29 15:56:57","publishedOnDateReadable":"September 29th, 2025"},"versionCreatedAt":"2025-03-31 14:19:02","video":"","vorDoi":"10.1007/s11696-025-04382-0","vorDoiUrl":"https://doi.org/10.1007/s11696-025-04382-0","workflowStages":[]},"version":"v1","identity":"rs-6166131","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6166131","identity":"rs-6166131","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}