An innovative technique for sensitive detection of carcinoembryonic antigen as a cell lung cancer marker

An innovative technique for sensitive detection of carcinoembryonic antigen as a cell lung cancer marker | 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 Short Report An innovative technique for sensitive detection of carcinoembryonic antigen as a cell lung cancer marker Jianjun Tang, ZhiQian Zhao, Yuxuan Xing, Sicong Jiang, Longhua Sun This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6891104/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Oct, 2025 Read the published version in Microchimica Acta → Version 1 posted 4 You are reading this latest preprint version Abstract Herein, a chemiluminescent (CL) aptasensor was proposed constructed by aptamer-aptamer bioconjugate for ultrasensitive determination of CYFRA21-1 as a lung cancer biomarker. The sensing mechanism was based on the incorporation of luminol into a cobalt-based metal–organic framework (MOF), also called ZIF-67, to generate a composite of the luminol@ZIF-67. And then, perceptional complex was modified with CYFRA21-1 specific aptamer (luminol@ZIF-67-apt) as signal probe. From this, ssDNA was immobilized on PVP/NiO nanocomposite which had been functionalized, allowing hybridization with the aptamer by complementary base pairing. In the presence of CYFRA 21-1, the target-CYFRA 21-1/luminol@ZIF-67-apt/PVP/NiO-ssDNA sandwich-like ternary complex could be assembled and induced the generation of intense CL emission. Under the optimized conditions, the sensor showed a broad linear detection ranging from 10 ⁻5 to 10⁴ ng/mL, with a detection limit (LOD) of 3.7 × 10 ⁻ ⁵ ng/mL. Reproducibility (RSD = 3.81%) and long-term stabilities was achieved, where the signal was respectively retained to 98.22 and 97.87% over 20 and 33 days. The recovery experiments in spiked human serum samples were in the range from 96.00% to 99.30%, and RSDs were varied from 3.41% to 4.07%, indicating that the aptasensor had good stability and good potential in complex biological matrices. Such a strategy holds a great promise for ultrasensitive detection of protein biomarkers for clinical bio-analysis. Chemiluminescence Cell lung cancer Cytokeratin 19 fragment Cobalt-based metal-organic framework Polyvinyl Pyrrolidone/Nickel Oxide Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction After heart disease, cancer was the second most common cause of mortality globally. One of the most common neoplasms is lung cancer, which often has a bad prognosis because most patients are diagnosed as having advanced or metastatic illness. Early detection and treatment can also prevent the disease from progressing, reducing the risk of complications and improving long-term health. There are several approaches to detecting cancer such. Various methods for cancer diagnosis, such as imaging tests, biopsy, lab tests, molecular diagnostics and physical examination, have been developed. However, these methods often face limitations such as lack of funding and awareness, lack of time and availability, invasiveness, expensive cost, and the need for specialized equipment and personnel which are the main global barriers to academic clinical cancer research. Therefore, the creation of accessible, affordable, and non-invasive sensor detection devices is essential for the accurate diagnosis of cell lung cancer as well as many other cancer types. Between these diagnostic methods, biomarkers have been exhibited remarkable attention because of their potential for early detection and monitoring of cancer. Proteins, DNA & RNAs, compounds, exosomes, and other biological molecule kinds and structures are examples of biomarkers. It can diagnose tumors early by seeing these biological warning indicators, which enhances treatment results for patients' quality of life. Lung cancer tumor indicators include cytokeratin 19 fragment (CYFRA 21 − 1), neuron-specific enolase (NSE), carcinoembryonic antigen (CEA), carcinoma of squamous cells antigen (SCC), and pro-gastrin-releasing peptide (proGRP). According to studies, of the five markers used to detect cell lung cancer, serum CYFRA 21 − 1 had the highest diagnostic sensitivity and accuracy [ 1 , 2 ]. Numerous methods, including electrochemical, chemiluminescent, colorimetric, immunoradiometric, and fluorescent techniques, are now used in the biosensing field to diagnose the biomarkers. Among these detection methods, chemiluminescent aptamer sensors stand out because of their high specificity, stability, sensitivity, and rapid response time. Chemiluminescence, characterized by light emission from a chemical reaction, offers notable advantages such as high sensitivity, low detection limits, wide linear dynamic ranges, simplicity, affordability, and rapid response. Integrating nanostructures greatly enhances the stability, selectivity, and sensitivity of chemiluminescent aptamer sensors for cancer biomarkers due to providing a large surface area for biomolecule interactions and improving signal transduction. Polyvinyl pyrrolidone (PVP) and nickel oxide (NiO) nanostructures are particularly advantageous due to their significant electrical conductivity, stability and biocompatibility. PVP shows notable stabilizing properties, preventing agglomeration and ensuring uniform size distribution of nanoparticles [ 3 ]. Researchers have also taken notice of PVP because to its unique characteristics, which include its inexpensive cost and good dielectric nature [ 4 ]. In addition, NiO nanostructures are known for its high thermo-chemical stability and low toxicity, making it a safe and reliable material for biosensing devices [ 5 ]. Furthermore, studies have been suggested that the developed MOF-based aptasensors such as ZIF-67 serve as highly sensitive and specific platforms for sensing cancer biomarkers with an extremely low limit of detection. Chemiluminescent detection is made easier by ZIF-67's huge surface area and porosity, which give a large number of active sites [ 6 ]. Furthermore, ZIF-67's hydrophobicity guarantees stability over an extended period of time in aqueous environments, making it ideal for usage in aptamer sensors. On the other hand, the PVP/NiO nanocomposite, conjugated with ZIF-67 offers a highly sensitive and stable platform for the development of chemiluminescent aptasensors. The large surface area of ZIF-67 and PVP/NiO nanocomposite can allows for an abundant number of aptamer molecules to be immobilized, increasing the likelihood of antigen molecule capture. The use of ZIF-67, in combination with PVP/NiO nanocomposites, provide a synergistic effect, leading in an aptasensor that is highly stable, specific and sensitive. In present work, an innative chemiluminescent aptasensor based on PVP/NiO-ssDNA/luminol@ZIF-67-apt was developed for sensitive and specific CYFRA 21 − 1 detection. The novelty of current study lies in the integration of ZIF-67 with PVP/NiO nanocomposite for modification chemiluminescent aptasensor that it can provide a highly selective, sensitive, and rapid diagnostic tool for early lung cancer detection. 2. Experiments 2.1. Chemicals and Reagents Nickel(II) acetate tetrahydrate, polyvinylpyrrolidone (PVP, 99%), sodium hydroxide (NaOH, 98%), cobalt(II) nitrate hexahydrate (Co(NO 3 ) 2 ·6H 2 O, 98%), 2-methylimidazole (99%), luminol and glutaraldehyde (25%) were purchased from Sigma-Aldrich, USA. Ethylene glycol (99.8%) and acetone (99.5%) were sourced from Merck, Germany. For the experiment, Sangon Biotech Co., Ltd. provided all of the nucleotide sequences. Thermo Fisher Scientific in the United States supplied the phosphate-buffered saline solution (PBS, pH 7.4). CYFRA 21-1 (a human cytokeratin 19 protein) was acquired from Abcam (Massachusetts, USA). Deionized water (99.9%) was obtained from Millipore. For synthesis of PVP/NiO [7], briefly, Ni(CH 3 COO) 2 ·4H 2 O solution ( 0.5 g) was dissolved in of ethylene glycol (20.0 mL) in a round flask (250 mL). Then, PVP (0.1 g) was added to the solution and stir magnetically until the PVP is fully dissolved. The solution was then heated to 150°C after a condenser was added to the system, followed by addition NaOH (0.2 g) to the solution and stirred vigorously to change the solution color to a dark precipitate. A steady colloidal dispersion was achieved after two hours of magnetic stirring at 150°C. Acetone (50.0 mL) was added to the dispersion in order to separate the dispersed black PVP/NiO nanoparticles. It was followed by centrifugation at 1000 rpm for 10 minutes. Afterwards, the obtained black solid was washed three times with acetone, and dry at room temperature. To prepare PVP/NiO-ssDNA, of PVP/NiO (30 mg) was shaken in a solution of glutaraldehyde (2 mL, 25%) for 100 minutes. Afterwards, ssDNA (10 mL, 10 µM) was added to the mixture, which then shaken for overnight to obtain PVP/NiO-ssDNA. For synthesis of ZIF-67 [8], briefly, Co(NO 3 ) 2 ·6H 2 O (0.9 g) was ultrasonically added in deionized water (5 mL). Then, the solution was mixed with 2-methylimidazole aquatic solution (40 mL, 300 mg/mL). After 6 hours magnetic stirring, the resulted purple mixture was centrifuged at 2000 rpm for 10 minutes. After that, the resulted purple precipitate was washed three times with deionized water and methanol, respectively, and then dried in an oven at 50°C. For the preparation of luminol@ZIF-67,luminol solution (10 mL, 5 mM) was added in NaOH (0.1 M). After 5 minutes magnetic stirring, the obtaned solution was mixed with ZIF-67 solution produced at ethanol (40 mL, 5 mg/mL). Following five hours of magnetic stirring at ambient temperature, the purple liquid was centrifuged for five minutes at 2000 rpm in order to extract supernatant. After that, the purple precipitate was dried at 65°C for 90 minutes to create luminol@ZIF-67 powder. The luminol@ZIF-67 powder is good for several months. To modify Luminol@ZIF-67-apt conjugates [9], breifly, 300 mg of synthesized luminol@ZIF-67 was dispersed in PBS (pH 7.4). Then, glutaraldehyde (2 mL, 25%) was added to mixture to activate the amino groups of luminol@ZIF-67. After that, the mixture was shaked for 2 hours. Subsequently, CYFRA 21-1 apt (10 mL, 5 µM) was added and the solution was stirred for 10 hours. The mixture was then centrifuged at 1000 rpm for 12 minutes to obtain luminol@ZIF-67-apt. Finally, the precipitates were dispersed for later use in buffered PBS (pH 7.4). Scanning Electron Microscope (SEM, JEOL Ltd.: JSM-IT510) was used for morphological studied of samples. The Dynamic-Light-Scattering(DLS) analysis was conducted on determining the size of nanoparticles using a Zetasizer Nano ZS (Malvern Panalytical, UK). Chemical structure was studied using Agilent Cary 630 FTIR (Agilent, USA). An Agilent Technologies Cary 630 Fourier transform infrared (FTIR)-ATR was used to identify the material's functional groups and chemical bonds (Agilent, USA). A fully automated area measurement and porosimetry analyser was used to determine the samples' specific surface area. Electrochemical experiments were performed with a VSP-300 (BioLogic, France) electrochemical workstation. UV-vis absorption spectra were obtained using a using an Evolution 300 Security UV-Vis Spectrophotometer (Thermo Fisher Scientific). Fluorescence measurements were conducted on a FluoroMax-4 (HORIBA Scientific, Japan) Fluorescence spectrometer. The wavelength measurements of the chemiluminescence system were obtained by adding a filter to a fluorescent device. A model of the MPI-F FIA-CL system was used for the CL measurements. Human Cytokeratin 19 Fragment Antigen 21-1 (CYFRA21-1) ELISA Kit (Abbexa Ltd., UK) was used for analyzing the real samples. trochemical experiments were performed with a CYFRA 21-1 was involved three steps. The first step was the participation of PVP/NiO-ssDNA/luminol@ZIF-67-apt. The second step was the addition of different concentrations CYFRA 21-1 to colorimetric tube holding the similar quantity of PVP/NiO-ssDNA/luminol@ZIF-67-apt and incubated for 20 minutes that the PVP/NiO-ssDNA/ luminol@ZIF-67-apt specifically recognizes CYFRA 21-1. The final step was the addition the supernatant collected in second step and transfer into the specimen tubes. The specimen and NaOH flowed through chief pump, while H 2 O 2 and PBS flowed through the auxiliary pump. Then, chemiluminescence analyzer was used for measurement the chemiluminescence intensity (I). The flow injection chemiluminescence analyzer system's schematic diagram is displayed in Scheme S1A. 3. Result and discussion In present study, PVP/NiO-ssDNA/luminol@ZIF-67-apt was used for CYFRA 21 − 1 recognition. Magnetic separation may be facilitated by the NiO component. The magnet pulled PVP/NiO-ssDNA/luminol@ZIF-67-apt to the bottom of a colorimetric tube. In antigen presence, luminol@ZIF-67-apt was separated from PVP/NiO-ssDNA, then bind directly to the antigen through its specific aptamer. PVP/NiO-ssDNA acts as a supporting matrix for the luminol@ZIF-67-apt, potentially plays a role in stabilty of system and amplify the detection signal. PVP serves as a stabilizing agent, improves the structural integrity of the NiO NPs and ssDNA. The PVP/NiO-ssDNA composite acts a a supportive matrix for luminol@ZIF-67-apt, enhancing its stability and facilitating its interaction with the target. PVP is also biocompatible and reduce the risk of adverse reactions and ensuring the system's safety. The presence of NiO NPs can potentially promot the chemiluminescence signal because it can enhance the catalytic activity in chemical reactions, and oxidation of luminol. Furthermore, aptamers serve as a recognition component for the lung cancer indicator antigen in addition to encapsulating luminol [ 10 ]. The ZIF-67 framework can be functionalized with targeting moieties, such as aptamers, to enable targeted delivery of luminol to specific cells or tissues [ 11 ]. The ZIF-67 has been used for encapsulating luminol molecules because of its structure which composed of cobalt(II) ions coordinated to 2-methylimidazole linkers, generating a sodalite topology with a large cavity size [ 12 ]. The ZIF-67 structure contains cavities or pores that can encapsulate guest molecules, such as luminol [ 13 ], and acts as a protective shell for luminol, shielding it from the environment and preventing premature oxidation [ 14 ]. Furthuremore, the ZIF-67 framework can control the release of luminol, ensuring that it is only released when needed, such as in the presence of the target antigen [ 9 ]. Luminol as a chemical fluorescent molecule is highly sensitive to light and incompatible with peroxidase activity. When H 2 O 2 is present, it can transform into an excited state and release intense luminosity [ 15 ]. The luminol encapsulated ZIF-67 structure can serves as the recognition element and the signal source. Upon reaction with appropriate reagents in the presence of PVP/NiO-ssDNA composite, ZIF-67 framework and H₂O₂, the luminol creates a chemiluminescent signal, the intensity of which is attributed to the antigen concentration. The encapsulation of Luminol molecules within the cavities of the ZIF-67 framework protects luminol from premature oxidation, improves its stability and prolongs luminol shelf life. In addition, ZIF-67 is biocompatible and possess a large surface area, which provide a large amount of luminol to be encapsulated, rising the sensitivity of the detection system. On the other hand, H 2 O 2 is analytically the most useful oxidant of luminol and It is the end result of numerous cellular oxidation processes, and these processes can be readily connected to optical detection [ 10 ]. Scheme S1B shows the schematic diagram of the experiment of chemiluminescent aptasensor. SEM images of luminol@ZIF-67 show Fig. 1 A. The images exhibit a uniform rhombic dodecahedral shape, which obviuosly suggest that the presence of luminol did not interfere with the formation of ZIF-67 nanoparticles. The average size of the luminol@ZIF-67 nanoparticles is about 500 nm. Figure 1 B shows results of size disparity of dynamic light scattering (DLS) of ZIF-67 synthesized and luminol@ZIF-67 that it is further confirmed by the avarages size of nanostructure and size distribution. Figure 1 C shows the structure information and ZIF-67's crystal phase and luminol@ZIF-67 which studied by XRD analysis. As can be observed, both the ZIF-67 and luminol@ZIF-67 samples have displayed distinctive MOF diffraction peaks due to ptresence of the chractristic peaks in the both XRD patterns at 7.19°, 10.41°, 12.68°, 14.72°, 16.42°, 18.05°, 22.07°, 24.51°, 25.57°, 29.58°, 31.31°, 32.50°, and 43.12° correspondig to (011), (002), (112), (022), (013), (222), (114), (233), (134), (044), (244), (235), and (100) difraction planes that The diffraction peaks closely match the standard specimen card (JCPDS card no. 00-062-1030), suggesting that ZIF-67 has a more complete crystal structure and is more pure [ 16 ]. The positions and intensities of the luminol@ZIF-67 diffraction peaks are found to be comparable to those of ZIF-67. These data corroborate the good crystallinity with luminol@ZIF-67 after luminol loading and demonstrate that ZIF-67 had well integrated within the synthesized composite, which is in line with the SEM results. The FT-IR spectrum of luminol, ZIF-67, with luminol@ZIF-67 are displayed in Fig. 1 E. The characteristic absorption spectrum of luminol, which have their centers at 1620 cm − 1 and 3400 cm − 1 , are seen in the luminol@ZIF-67 and luminol FT-IR spectra. These bands are attributed to the vibrations that stretch for C = O and N-H as of luminol's primary amine group (-NH2) [ 17 , 18 ]. ZIF-67and luminol@ZIF-67 FT-IR spectrum demonstrate the strong bands of absorption at 425 cm − 1 , 990 cm − 1 , and 1573 cm − 1 that are ascribed to the bending stretching vibrations of Co–N (metal-ligand), C–N,andC = N [ 19 , 20 ]. These observation indicate the successful encapsulation of luminol within ZIF-67. The luminol@ZIF-67 XPS survey spectra are revealed in Fig. 2 A. As can be seen from the detected C 1s, N 1s, O 1s, and Co 2p peaks, the XPS survey suggests the existence of C,N,O, and Co. Cobalt atoms in Co–N bonds are represented by the detected peaks on 785.8 eV and 801.2 eV in the high-resolution XPS spectrum of Co2p in Fig. 2 B [ 21 ], while Co 2p3/2 and Co 2p1/2 of Co 2+ are represented by the peaks at 781.0 eV and 796.7 eV, respectively, and can be credited to cobalt atoms in Co–O bonds [ 22 ]. The carbon atom within the –C–NH2 group and the –C = O– group from luminol are identified by peaks centered in 286.2 eV and 288.3 eV, respectively, in the high-resolution XPS spectrum of C 1s in Fig. 2 C [ 23 , 24 ]. The presence of carbon in the C–N, O–C–NH, and –C = N– groups from luminol is attributed to the peaks that appear at 399.31 eV, 399.75 eV, and 400.45 eV in the high-resolution XPS spectrum of N 1s in Fig. 2 D, respectively. The peak at 398.92 eV is attributed to N–Co [ 23 , 24 ]. These findings further demonstrate that luminol was successfully encapsulated within ZIF-67. Figure S1 A depicts the XRD pattern of PVP, NiO and PVP/NiO nanocomposite. XRD pattern of PVP shows two broad peaks aproximately 2θ of 11.5° and 21.2° and these peaks are observed in PVP/NiO at 2θ of 11.6 and 21.5°. It is found that XRD pattern of NiO and PVP/NiO samples samples clearly exhibit the diffraction peaks of (111),(200),(220),(311),and (222) plane, indexed into face-centred-cubic (fcc) structured NiO (JCPDS card no. 00-047-1049) [ 25 ]. XRD patterns of PVP/NiO nanocomposite shows the diffraction peaks that they are low and broad due to the PVP anchoring in nanocomposite. Figure S1 B depicts the FT-IR spectra of PVP and PVP/NiO nanocomposite. Both spectra show a broad band at about 3445 cm -1 attributed to O-H group bending vibration and adsorbed water molecules [ 26 ]. As observed, a band located at 1675 cm -1 is related to the carbonyl (C = O) stretching vibration of PVP [ 27 ]. Around 2950 cm − 1 is where the band associated with PVP's CH 2 asymmetric vibration of stretching is located [ 28 ], and the band at around 1440 cm -1 is ascribed to the vibration of heterocyclic in PVP [ 29 ]. A strong peak at 470 cm -1 in PVP/NiO is attributed to the Ni-O stretching mode [ 30 ]. These characteristic peaks demonestrate the successful synthesis of PVP/NiO nanocomposite. The peroxidase-like activity of luminol@ZIF-67 was demonestrated through the catalytic oxidation of H 2 O 2 , as exhibited in Fig. 3 A. The peak of absorption at 652 nm was used to study the catalytic activity [ 31 ]. Results show that luminol@ZIF-67 reveals a maximum absorbance at 652 nm, asociated with the oxidation of H 2 O 2 , demonestrating the catalytic activity of Luminol@ZIF-67 is related to presence of luminol. Co-imidazole MOF (ZIF-67) was shown in previous research to be an efficient CL catalyst for the luminol-H2O2 CL reaction [ 32 , 33 ]. Several substances have been included to the reaction while maintaining equivalent luminol concentration in order to further elucidate the catalytic activity for luminol@ZIF-67. Imidazole may enhance the catalytic activity on peroxidase-like activity, as seen by the control studies in Fig. 3 B, which demonstrate that the catalytic activity within the existence of 2-methylimidazole in solutions is greater than that of luminol alone. Moreover, Michaelis-Menten constant (K m ) was calculated using the Lineweaver-Menten plot [ 34 ]. The relatively small K m values for H 2 O 2 (0.078 mM) that lower than the Km of horseradish peroxidase [ 35 ], nanostructured polymer membrane [ 36 ], NiO thin film [ 37 ] and NiO nanoparticles [ 38 ] demonestrate an increased affinity between H 2 O 2 and luminol@ZIF-67, indicating higher affinity of luminol@ZIF-67 for H 2 O 2 . Additionally, the catalytic efficiency of ZIF-67 and luminol@ZIF-67 in an alkaline solution was examined. The catalytic efficiency of luminol@ZIF-67 is demonstrated in Fig. 3 C to be greater than roughly six times that of luminol or ZIF-67 alone in the CL system. This enhancement is asociated with the tendency of luminol to form dimers in aqueous solution, a problem that was mitigated by incorporating luminol into ZIF-67 [ 39 – 41 ]. Therefore, the synergistic catalytic properties of both luminol and ZIF-67 can promote the luminol@ZIF-67 catalytic activity. To examine local electron conversion resulted from catalytic Luminol@ZIF-67 activity in the CL reaction by activating H 2 O 2 , phenylhydrazine and thiourea employed to assess the existence of • O 2 − and • OH radicals [ 42 ]. Figure 3 D displays that the existence of phenylhydrazine into the sensor structure led to decrease the CL signal, revealing that luminol@ZIF-67 enhance the creation of • O 2 − . Results show that thiourea as scavenger in • OH effectively inhibited the CL signal, implying that • OH is an essential intermediate in the CL system catalyzed by luminol@ZIF-67. Furthermore, Fig. 3 E indicates that the CL emitter corresponds to the excited-state 3-aminophthalate anion, as the highest emission wavelength for luminol around 440 nm is the same for luminol@ZIF-67-hemin [ 43 ]. Furthermore, Fig. 3 F compares the CL signal's level in air, N 2 , and O 2 environments, demonstrating the critical role that oxygen plays in our CL aptasensor. For optimization the conditions for CL aptasensor the effect of ratio of PVP/NiO-ssDNA to luminol@ZIF-67-apt, antigen incubation time, pH of the buffer, NaOH concentration, H 2 O 2 concentration and flow rate on sensitivity of the proposed aptasensor were investigated, and results show in Figure S2, respectively. Results indicate maximum signal of CL aptasensor is obtained in equal ratio of PVP/NiO-ssDNA to luminol@ZIF-67-apt, 20 minutes for antigen incubation time, buffer solution with pH of 7.4, 0.05 M NaOH of, 0.15 M H 2 O 2 , the main pump's flow rate was 25 r/min, respectively. Therefore, these conditions were selected as the optimal aptasensor. Under optimized condition, the obtained CL aptamer signal for various concentrations of the target antigen is shown in Fig. 4 A. As seen, there is good linearity with increasing concentrations of the target antigen and resulted signal of aptasensor (PVP/NiO-ssDNA/luminol@ZIF-67-apt). The analytical performance of the aptasensor was assessed by measuring the resulted signal and observed that the signal of aptasensor is strongly correlated with the target antigen concentration (R 2 = 0.99946) with equation: I = 6109.2179 + 431.9149 Log C CYFRA 21−1 (Fig. 4 B). The limit of detection (LOD) reaches as low as 3.7×10 − 5 ng/mL based on calculating the 3σ/S criterion (where ‘S’ is the slope of the calibration plot and σ is the standard deviation of the blank). These observations indicate the simplified detection of lung cancer marker via the generation of CL signal using PVP/NiO-ssDNA/luminol@ZIF-67-apt aptasensor. The clinically relevant serum concentration of CYFRA 21 − 1 in lung cancer patients typically ranges from 1 to 10 ng/mL, with levels above 3.3 ng/mL considered indicative of malignancy. The aptasensor developed in this study exhibits a wide linear detection range from 10⁻⁵ to 10⁴ ng/mL and an ultralow detection limit of 3.7 × 10⁻⁵ ng/mL, allowing for accurate detection of even trace levels of CYFRA 21 − 1. This confirms its practical applicability for clinical screening and early-stage diagnosis, providing a robust and sensitive method for monitoring lung cancer biomarkers in patient samples. The comparison between current study and with previously reported CYFRA 21 − 1 sensors is presented in Table 1 . It demonstrates that the present CL aptamer nanoprobe as selective sensor offers notably sensitivity and comparable or better performance compared to some previously reported sensors. Table 1 Comparision between the current study and with previously reported CYFRA 21 − 1 sensors. Method Reagent LOD (ng/mL) Linear range (ng/mL) Ref. CL aptasensor PVP/NiO-ssDNA/luminol@ZIF-67-apt 3.7×10 − 5 10 − 5 to 10 4 This work AlphaScreen AlphaLISA kit based on antibody coated on AlphaLISA acceptor beads and donor beads coated with streptavidin 0.08 0.08 to 500 [ 44 ] ECL tris(2,29-bipyridyl)ruthenium (II) complex 0.2 2 to 30 [ 45 ] ECL Magnetic beads loaded with conductive carbon black 1.14×10 − 4 10 − 3 to 10 2 [ 46 ] ECL Antibody/3D graphene @ Au NPs/GCE 0.1 0.25 to 800 [ 47 ] ECL Antibody/3D graphene/chitosan/glutaraldehyde/GCE 0.043 0.1 to 150 [ 48 ] ECL Antibody/toluidine blue /AuNPs@MoS 2 @Ti 3 C 2 T x 3×10 − 6 0.5 to 50 [ 49 ] ECL Antibody/3-mercaptopropionic 9.08×10 − 6 10 − 3 to 10 3 [ 50 ] ECL MoOx quantum dots 3×10 − 4 10 − 3 to 350 [ 51 ] ECL Toluidine blue/AuNPs/Ti 3 C 2 T x -MXene 10 − 4 5 to 10 [ 52 ] EIS Antibody/amino terminal groups of PTNH 2 polymer 4.7×10 − 6 3×10 − 6 to 0.09 [ 53 ] EIS Antibody/Au NPs@CMK-3@CMWCNTs 3×10 − 4 5×10 − 4 to 10 2 [ 54 ] IRA enzyme immunoassay 3.5 ---- [ 55 ] REI amino-Antibody/Ag/BSA/Antibody/MB/CdTe/MoS 2 /GCE --- 10 − 3 to 10 4 [ 56 ] UV-Vis Antibody/AuNPs)/reduced graphene oxide/indium tin oxide 20 25×10 − 5 to 20 [ 57 ] LSPR Antibody/Au nanorods 0.84 0.496 to 48.4 [ 58 ] SERS Au @Ag NRs 8.9×10 − 4 10 − 3 to 10 [ 59 ] CL: Chemiluminescent; ECL: Electrochemiluminescent; EIS: Electrochemical impedance spectroscopy; IRA: Immunoradiomentric assay; REI: Ratiometric Electrochemical immunoassay; UV-Vis: UV-Visible spectrophotometer; LSPR: Localized Surface Plasmon Resonance; SERS: Surface-enhanced Raman scattering The consistency of the proposed aptasensor was examined by analyzing five aptasensors fabricated under same conditions and at a CYFRA 21 − 1 concentration of 1 ng/mL. Figure 5 a shows that the RSD is found to be 3.71%, indicating the reproducibility of the aptasensor. Another important property attribute of the proposed aptasensor is its long-term stability. It was evaluated during a thirty-three-day period. The results demonstrated in Fig. 5 B reveal that initial resistances are maintained at 98.22% and 97.87% after 20 and 33 days, respectively, indicating appropriate stability. The specificity of the fabricated aptasensor was also evaluated under optimal conditions. The aptasensor’s response to potential interferences such as neuron-specific enolase (NSE), carcinoembryonic antigen (CEA), alpha fetal protein (AFP), squamous cell carcinoma antigen (SCC), immunoglobulin G (IgG) and BSA were recorded and juxtaposed with the response of the designed aptasensor. Figure 5 C shows that the CL signal of the fabricated aptasensor to CYFRA 21 − 1 remarkably surpassed that of other compounds, highlighting that the aptasensor possesses a distinctive recognition capability towards CYFRA 21 − 1 and corroborates its acceptable specificity. To examine the practicability of the aptasensor for CYFRA 21 − 1, recovery tests were conducted which involved the assay of four different concentrations (0.50, 1.00, 10.00 and 20.00 ng/mL of CYFRA 21 − 1) in a human serum sample, using the standard addition method. Table 2 summarizes the obtained results and illustrates to acceptable of the recoveries in range of 96.00 to 99.30% and RSD value from 3.41–4.07%. the obtained results of the commercially available ELISA assay are presented in Table 2 . As found, the responses of the proposed aptasensor are close those of the ELISA assay, suggesting that the suggested aptasensor can detect CYFRA 21 − 1 in actual samples with sufficient accuracy and validity. Moreover, it demonstrates a potential for precise clinical applications and pharmaceutical researches. Table 2 The result of CYFRA 21-1detection in human serum samples. Spiked (ng/mL) Present aptasensor ELISA Relative error (%) found (ng/mL) Recovery (%) RSD (%) found (ng/mL) Recovery (%) RSD (%) 0.50 0.48 96.00 4.07 0.49 98.00 3.66 2.04082 1.00 0.97 97.00 3.41 0.98 98.00 4.14 1.02041 10.00 9.93 99.30 3.84 9.92 99.20 3.73 -0.10081 20.00 19.85 99.25 4.05 19.82 99.10 4.15 -0.15136 4. Conclusion A CL aptasensor based on an aptamer conjugate was created in this study to identify CYFRA 21 − 1 as a biomarker for lung cancer. This CL aptasensor offers several advantages: (1) The use of luminol@ZIF-67-apt as probe remarkably improved sensitivity in the CL system, (2) luminol@ZIF-67 effectively addresses the issue of luminol dimer formation in solution, thereby dramatically improving the CL signal, and (3) The incorporation of PVP/NiO-ssDNA effectively minimizes background interference during CYFRA 21 − 1 detection. These benefits demonstrated the sensing platform's promise in intricate clinical settings, as demonstrated by the accurate assessment of CYFRA 21 − 1 amounts in actual samples. An encouraging substitute of CYFRA 21 − 1 detection for human serum samples was offered by the developed aptasensor. The aptasensor showed significant performance characteristics, including a low detection limit (3.7×10 − 5 ng/mL), broad linear range (10 − 5 to 10 4 ng/mL), remarkable reproducibility, and long-term stability (maintaining initial resistance at 97.87% after 33 days). Moreover, the aptamer strategy improves the selectivity of the aptasensor, reducing the likelihood of false positives and ensuring reliable detection. Future work could focus on further optimizing the aptasensor design and expanding its applications in the field of cancer diagnostics in clinical applications. Declarations Funding The authors did not receive support from any organization for the submitted work. References Wieskopf B, Demangeat C, Purohit A, Stenger R, Gries P, Kreisman H, Quoix E (1995) Cyfra 21-1 as a biologic marker of non-small cell lung cancer. Evaluation of sensitivity, specificity, and prognostic role. Chest 108:163-169. Muraki M, Tohda Y, Iwanaga T, Uejima H, Nagasaka Y, Nakajima S (1996) Assessment of serum CYFRA 21‐1 in lung cancer. Cancer: Interdisciplinary International Journal of the American Cancer Society 77:1274-1277. 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Supplementary Files Scheme S1 and Figure S1 are not available with this version Additional Declarations No competing interests reported. Supplementary Files Graphicalabstract.jpg Cite Share Download PDF Status: Published Journal Publication published 30 Oct, 2025 Read the published version in Microchimica Acta → Version 1 posted Editorial decision: Revision requested 18 Jun, 2025 Editor assigned by journal 16 Jun, 2025 Submission checks completed at journal 16 Jun, 2025 First submitted to journal 13 Jun, 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6891104","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":472960532,"identity":"1f24a7c5-5390-424e-87de-a43f2aaae65b","order_by":0,"name":"Jianjun Tang","email":"","orcid":"","institution":"The First Affiliated Hospital of Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Jianjun","middleName":"","lastName":"Tang","suffix":""},{"id":472960533,"identity":"55889d58-5658-4e1c-b837-219772eee82f","order_by":1,"name":"ZhiQian Zhao","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University, Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"ZhiQian","middleName":"","lastName":"Zhao","suffix":""},{"id":472960534,"identity":"3468d239-84b0-4394-92e5-6cc7d2acb275","order_by":2,"name":"Yuxuan Xing","email":"","orcid":"","institution":"The First Affiliated Hospital of Soochow University, Suzhou University","correspondingAuthor":false,"prefix":"","firstName":"Yuxuan","middleName":"","lastName":"Xing","suffix":""},{"id":472960535,"identity":"6808fdf6-9a3d-46be-986d-f65c8f91a098","order_by":3,"name":"Sicong Jiang","email":"","orcid":"","institution":"The First Affiliated Hospital of Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Sicong","middleName":"","lastName":"Jiang","suffix":""},{"id":472960536,"identity":"9583e2e7-6ec3-41e6-98c7-d66d1c08e6f3","order_by":4,"name":"Longhua Sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvklEQVRIiWNgGAWjYHACxsMMBjZy/MzMhx8QrecwQ0WasWQ7W5oBCVrOHE7ccJ5HQYIo5fIzcg8cLmxLS9x8mIfBgKHGJpqgFoMbeQmHZ7bZGG87zHvgAcOxtNwGglokcgwO87alyW47zJdgwNhwmLAW+RlgLYcZNzfzGEgQpYXhBlALz5nDihuYidVicOYNUAswkCUOAwM5gRi/yLfnGD7mAUVl/+HDDz7U2BDhMBSQQJryUTAKRsEoGAW4AAD8/0ESNx21/gAAAABJRU5ErkJggg==","orcid":"","institution":"The First Affiliated Hospital of Nanchang University","correspondingAuthor":true,"prefix":"","firstName":"Longhua","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2025-06-14 00:38:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6891104/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6891104/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00604-025-07590-3","type":"published","date":"2025-10-30T15:56:56+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85996318,"identity":"88849aeb-fffc-4c40-acd2-f5adeee14903","added_by":"auto","created_at":"2025-07-04 06:17:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1447769,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Luminol@ZIF-67 SEM pictures. (B) Synthetic ZIF-67 and luminol@ZIF-67 size discrepancy dynamic light scattering(DLS) results ZIF-67and luminol@ZIF-67 XRD patterns (C) ZIF-67, luminol, and (D) luminol@ZIF-67 FT-IR spectrum.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6891104/v1/396af1d47b2653d9aae02a4c.png"},{"id":85997080,"identity":"a2d278b7-c9f0-47f5-93e1-c6068b04667c","added_by":"auto","created_at":"2025-07-04 06:25:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1365862,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Luminol@ZIF-67 XPS survey spectra and high-resolution XPS spectrum (B) Co 2p, (C) C 1s, and (D) N 1s.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6891104/v1/42db2fedae39e1e8b70c5293.png"},{"id":85996314,"identity":"b51009ae-55ee-425e-b3fe-7ee61d60c925","added_by":"auto","created_at":"2025-07-04 06:17:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":652558,"visible":true,"origin":"","legend":"\u003cp\u003e(A) The absorption plots of catalytic reaction, (B) Relative activity of luminol, luminol+ZIF-67, luminol@ZIF-67, luminol+2-methylimidazole,luminol+Zn\u003csup\u003e2+\u003c/sup\u003e. (C) The CL spectra: hemin+luminol+H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, hemin+ZIF-67+H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, hemin+luminol@ZIF-67+H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, hemin+PBS+H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. \u0026nbsp;(D) The CL spectra: hemin+luminol@ZIF-67+H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, hemin+luminol@ZIF-67+H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e+phenylhydrazine, hemin+hemin@ ZIF-67+H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e+thiourea. (E) The CL spectra: luminol+H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, hemin+ZIF-67+H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. (F) The CL spectra: hemin+luminol@ZIF-67+H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e+O\u003csub\u003e2\u003c/sub\u003e,hemin+luminol@ZIF-67+H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e+air, hemin+luminol@ZIF-67+H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e+N\u003csub\u003e2\u003c/sub\u003e. luminol: 0.2 M; luminol@ZIF-67 :0.05 mg/mL; NaOH: 0.05 M; H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e: 0.15 M.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6891104/v1/ee1c2ef2f4615a95816148e6.png"},{"id":85997081,"identity":"9b84f4ed-942e-4888-b26a-b1bd74a7c808","added_by":"auto","created_at":"2025-07-04 06:25:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":663152,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of\u0026nbsp; (A) ratio of PVP/NiO-ssDNA to luminol@ZIF-67-apt, (B) antigen incubation time, (C) pH of the buffer, (D) NaOH concentration, (E) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration and (F) flow rate on signal of the proposed aptasensor in presence 0.1 M PBS containing 1 ng/mL CYFRA 21-1.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6891104/v1/f20810aa006fb69044184ac2.png"},{"id":85997079,"identity":"ae2a57d5-495b-454f-893d-1f4c09969e2b","added_by":"auto","created_at":"2025-07-04 06:25:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":175316,"visible":true,"origin":"","legend":"\u003cp\u003e(A) The response of the CL aptasensor to various concentrations of the target, and (B) The obtained CL intensity, which is proportional to the analyte concentration logarithm throughout a range of 10\u003csup\u003e-5\u003c/sup\u003e to 10\u003csup\u003e4\u003c/sup\u003e ng/mL.\u0026nbsp;\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6891104/v1/3da8fac7b435a0d5e5df8a08.png"},{"id":85996321,"identity":"e9ebb41e-e09d-4a82-a997-4575f7a09aab","added_by":"auto","created_at":"2025-07-04 06:17:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":181709,"visible":true,"origin":"","legend":"\u003cp\u003eResult of evaluation (A) the reproducibility for five fabricated aptasensor under the identical condition, (B) the long-term stability of the aptasensor aver 33 days, and (C) the specificity of the aptasensor in presence other compounds (20 ng/mL) in 0.1 M PBS containing 1 ng/mL\u003csup\u003e \u003c/sup\u003eCYFRA 21-1.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6891104/v1/991e3f20bdb88486e97f92c3.png"},{"id":95040662,"identity":"7e193d0e-ad95-4a87-9e73-21abcceb736e","added_by":"auto","created_at":"2025-11-03 16:10:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5869008,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6891104/v1/b7869058-3c08-4d01-88c5-117f99480440.pdf"},{"id":85996316,"identity":"1aeced6b-a81e-4e4c-b5fa-67ab4eae2346","added_by":"auto","created_at":"2025-07-04 06:17:16","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":137360,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6891104/v1/8717339f989f5e5cfccb6efc.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"An innovative technique for sensitive detection of carcinoembryonic antigen as a cell lung cancer marker","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAfter heart disease, cancer was the second most common cause of mortality globally. One of the most common neoplasms is lung cancer, which often has a bad prognosis because most patients are diagnosed as having advanced or metastatic illness. Early detection and treatment can also prevent the disease from progressing, reducing the risk of complications and improving long-term health. There are several approaches to detecting cancer such. Various methods for cancer diagnosis, such as imaging tests, biopsy, lab tests, molecular diagnostics and physical examination, have been developed. However, these methods often face limitations such as lack of funding and awareness, lack of time and availability, invasiveness, expensive cost, and the need for specialized equipment and personnel which are the main global barriers to academic clinical cancer research. Therefore, the creation of accessible, affordable, and non-invasive sensor detection devices is essential for the accurate diagnosis of cell lung cancer as well as many other cancer types.\u003c/p\u003e \u003cp\u003eBetween these diagnostic methods, biomarkers have been exhibited remarkable attention because of their potential for early detection and monitoring of cancer. Proteins, DNA \u0026amp; RNAs, compounds, exosomes, and other biological molecule kinds and structures are examples of biomarkers. It can diagnose tumors early by seeing these biological warning indicators, which enhances treatment results for patients' quality of life. Lung cancer tumor indicators include cytokeratin 19 fragment (CYFRA 21\u0026thinsp;\u0026minus;\u0026thinsp;1), neuron-specific enolase (NSE), carcinoembryonic antigen (CEA), carcinoma of squamous cells antigen (SCC), and pro-gastrin-releasing peptide (proGRP). According to studies, of the five markers used to detect cell lung cancer, serum CYFRA 21\u0026thinsp;\u0026minus;\u0026thinsp;1 had the highest diagnostic sensitivity and accuracy [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Numerous methods, including electrochemical, chemiluminescent, colorimetric, immunoradiometric, and fluorescent techniques, are now used in the biosensing field to diagnose the biomarkers. Among these detection methods, chemiluminescent aptamer sensors stand out because of their high specificity, stability, sensitivity, and rapid response time. Chemiluminescence, characterized by light emission from a chemical reaction, offers notable advantages such as high sensitivity, low detection limits, wide linear dynamic ranges, simplicity, affordability, and rapid response. Integrating nanostructures greatly enhances the stability, selectivity, and sensitivity of chemiluminescent aptamer sensors for cancer biomarkers due to providing a large surface area for biomolecule interactions and improving signal transduction.\u003c/p\u003e \u003cp\u003ePolyvinyl pyrrolidone (PVP) and nickel oxide (NiO) nanostructures are particularly advantageous due to their significant electrical conductivity, stability and biocompatibility. PVP shows notable stabilizing properties, preventing agglomeration and ensuring uniform size distribution of nanoparticles [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Researchers have also taken notice of PVP because to its unique characteristics, which include its inexpensive cost and good dielectric nature [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In addition, NiO nanostructures are known for its high thermo-chemical stability and low toxicity, making it a safe and reliable material for biosensing devices [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Furthermore, studies have been suggested that the developed MOF-based aptasensors such as ZIF-67 serve as highly sensitive and specific platforms for sensing cancer biomarkers with an extremely low limit of detection.\u003c/p\u003e \u003cp\u003eChemiluminescent detection is made easier by ZIF-67's huge surface area and porosity, which give a large number of active sites [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Furthermore, ZIF-67's hydrophobicity guarantees stability over an extended period of time in aqueous environments, making it ideal for usage in aptamer sensors. On the other hand, the PVP/NiO nanocomposite, conjugated with ZIF-67 offers a highly sensitive and stable platform for the development of chemiluminescent aptasensors. The large surface area of ZIF-67 and PVP/NiO nanocomposite can allows for an abundant number of aptamer molecules to be immobilized, increasing the likelihood of antigen molecule capture. The use of ZIF-67, in combination with PVP/NiO nanocomposites, provide a synergistic effect, leading in an aptasensor that is highly stable, specific and sensitive.\u003c/p\u003e \u003cp\u003eIn present work, an innative chemiluminescent aptasensor based on PVP/NiO-ssDNA/luminol@ZIF-67-apt was developed for sensitive and specific CYFRA 21\u0026thinsp;\u0026minus;\u0026thinsp;1 detection. The novelty of current study lies in the integration of ZIF-67 with PVP/NiO nanocomposite for modification chemiluminescent aptasensor that it can provide a highly selective, sensitive, and rapid diagnostic tool for early lung cancer detection.\u003c/p\u003e"},{"header":"2. Experiments","content":"\u003cp\u003e\u003cstrong\u003e2.1. Chemicals and Reagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNickel(II) acetate tetrahydrate, polyvinylpyrrolidone (PVP, 99%), sodium hydroxide (NaOH, 98%), cobalt(II) nitrate hexahydrate (Co(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO, 98%), 2-methylimidazole (99%), luminol and glutaraldehyde (25%) were purchased from Sigma-Aldrich, USA. Ethylene glycol (99.8%) and acetone (99.5%) were sourced from Merck, Germany. For the experiment, Sangon Biotech Co., Ltd. provided all of the nucleotide sequences. Thermo Fisher Scientific in the United States supplied the phosphate-buffered saline solution (PBS, pH 7.4). CYFRA 21-1 (a human cytokeratin 19 protein) was acquired from Abcam (Massachusetts, USA). Deionized water (99.9%) was obtained from Millipore.\u003c/p\u003e\n\u003cp\u003eFor synthesis of PVP/NiO [7], briefly, Ni(CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003e\u0026middot;4H\u003csub\u003e2\u003c/sub\u003eO solution ( 0.5 g) was dissolved in of ethylene glycol (20.0 mL) in a round flask (250 mL). Then, PVP (0.1 g) was added to the solution and stir magnetically until the PVP is fully dissolved. The solution was then heated to 150\u0026deg;C after a condenser was added to the system, followed by addition NaOH (0.2 g) to the solution and stirred vigorously to change the solution color to a dark precipitate. A steady colloidal dispersion was achieved after two hours of magnetic stirring at 150\u0026deg;C. \u0026nbsp;Acetone (50.0 mL) was added to the dispersion in order to separate the dispersed black PVP/NiO nanoparticles. It was followed by centrifugation at 1000 rpm for\u0026nbsp;10\u0026nbsp;minutes. Afterwards,\u0026nbsp;the obtained black solid was washed three times with acetone, and dry at room temperature. To prepare PVP/NiO-ssDNA, of PVP/NiO (30 mg) was shaken in a solution of glutaraldehyde (2 mL, 25%) for 100 minutes. Afterwards, ssDNA (10 mL, 10 \u0026micro;M) was added to the mixture, which then shaken for overnight to obtain PVP/NiO-ssDNA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor synthesis of ZIF-67 [8], briefly, Co(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO (0.9 g) was ultrasonically added in deionized water (5 mL). Then, the solution was mixed with 2-methylimidazole aquatic solution (40 mL, 300 mg/mL). After 6 hours magnetic stirring, the resulted purple mixture was centrifuged at 2000 rpm for 10 minutes. After that, the resulted purple precipitate was washed three times with deionized water and methanol, respectively, and then dried in an oven at 50\u0026deg;C.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the preparation of luminol@ZIF-67,luminol solution (10 mL, 5 mM) was added in NaOH (0.1 M). After 5 minutes magnetic stirring, the obtaned solution was mixed with ZIF-67 solution produced at ethanol (40 mL, 5 mg/mL). Following five hours of magnetic stirring at ambient temperature, the purple liquid was centrifuged for five minutes at 2000 rpm in order to extract supernatant. After that, the purple precipitate was dried at 65\u0026deg;C for 90 minutes to create luminol@ZIF-67 powder. \u0026nbsp;The luminol@ZIF-67 powder is good for several months.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eTo modify Luminol@ZIF-67-apt conjugates [9], breifly, 300 mg of synthesized luminol@ZIF-67 was dispersed in PBS (pH 7.4). Then, glutaraldehyde (2 mL, 25%) was added to mixture to activate the amino groups of luminol@ZIF-67. After that, the mixture was shaked for 2 hours. Subsequently, CYFRA 21-1 apt (10 mL, 5 \u0026micro;M) was added and the solution was stirred for 10 hours. The mixture was then centrifuged at 1000 rpm for 12 minutes to obtain luminol@ZIF-67-apt. Finally, the precipitates were dispersed for later use in buffered PBS (pH 7.4).\u003c/p\u003e\n\u003cp\u003eScanning Electron Microscope (SEM, JEOL Ltd.: JSM-IT510) was used for morphological studied of samples. The Dynamic-Light-Scattering(DLS) analysis was conducted on determining the size of nanoparticles using a Zetasizer Nano ZS (Malvern Panalytical, UK). Chemical structure was studied using Agilent Cary 630 FTIR (Agilent, USA). An Agilent Technologies Cary 630 Fourier transform infrared (FTIR)-ATR was used to identify the material\u0026apos;s functional groups and chemical bonds (Agilent, USA). A fully automated area measurement and porosimetry analyser was used to determine the samples\u0026apos; specific surface area. Electrochemical experiments were performed with a VSP-300 (BioLogic, France) electrochemical workstation. UV-vis absorption spectra were obtained using a using an Evolution 300 Security UV-Vis Spectrophotometer (Thermo Fisher Scientific). Fluorescence measurements were conducted on a FluoroMax-4 (HORIBA Scientific, Japan) Fluorescence spectrometer. The wavelength measurements of the chemiluminescence system were obtained by adding a filter to a fluorescent device. A model of the MPI-F FIA-CL system was used for the CL measurements. Human Cytokeratin 19 Fragment Antigen 21-1 (CYFRA21-1) ELISA Kit (Abbexa Ltd., UK) was used for analyzing the real samples.\u003c/p\u003e\n\u003cp\u003etrochemical experiments were performed with a CYFRA 21-1 was involved three steps. The first step was the participation of PVP/NiO-ssDNA/luminol@ZIF-67-apt. The second step was the addition of different concentrations CYFRA 21-1 to colorimetric tube holding the similar quantity of PVP/NiO-ssDNA/luminol@ZIF-67-apt and incubated for 20 minutes that the PVP/NiO-ssDNA/ luminol@ZIF-67-apt specifically recognizes CYFRA 21-1. The final step was the addition the supernatant collected in second step and transfer into the specimen tubes. The specimen and NaOH flowed through chief pump, while H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and PBS flowed through the auxiliary pump.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThen, chemiluminescence analyzer was used for measurement the chemiluminescence intensity (I). The flow injection chemiluminescence analyzer system\u0026apos;s schematic diagram is displayed in Scheme S1A.\u003c/p\u003e\n"},{"header":"3. Result and discussion","content":"\u003cp\u003eIn present study, PVP/NiO-ssDNA/luminol@ZIF-67-apt was used for CYFRA 21\u0026thinsp;\u0026minus;\u0026thinsp;1 recognition. Magnetic separation may be facilitated by the NiO component. The magnet pulled PVP/NiO-ssDNA/luminol@ZIF-67-apt to the bottom of a colorimetric tube. In antigen presence, luminol@ZIF-67-apt was separated from PVP/NiO-ssDNA, then bind directly to the antigen through its specific aptamer. PVP/NiO-ssDNA acts as a supporting matrix for the luminol@ZIF-67-apt, potentially plays a role in stabilty of system and amplify the detection signal. PVP serves as a stabilizing agent, improves the structural integrity of the NiO NPs and ssDNA. The PVP/NiO-ssDNA composite acts a a supportive matrix for luminol@ZIF-67-apt, enhancing its stability and facilitating its interaction with the target. PVP is also biocompatible and reduce the risk of adverse reactions and ensuring the system's safety. The presence of NiO NPs can potentially promot the chemiluminescence signal because it can enhance the catalytic activity in chemical reactions, and oxidation of luminol. Furthermore, aptamers serve as a recognition component for the lung cancer indicator antigen in addition to encapsulating luminol [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The ZIF-67 framework can be functionalized with targeting moieties, such as aptamers, to enable targeted delivery of luminol to specific cells or tissues [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The ZIF-67 has been used for encapsulating luminol molecules because of its structure which composed of cobalt(II) ions coordinated to 2-methylimidazole linkers, generating a sodalite topology with a large cavity size [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The ZIF-67 structure contains cavities or pores that can encapsulate guest molecules, such as luminol [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and acts as a protective shell for luminol, shielding it from the environment and preventing premature oxidation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Furthuremore, the ZIF-67 framework can control the release of luminol, ensuring that it is only released when needed, such as in the presence of the target antigen [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Luminol as a chemical fluorescent molecule is highly sensitive to light and incompatible with peroxidase activity. When H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is present, it can transform into an excited state and release intense luminosity [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The luminol encapsulated ZIF-67 structure can serves as the recognition element and the signal source. Upon reaction with appropriate reagents in the presence of PVP/NiO-ssDNA composite, ZIF-67 framework and H₂O₂, the luminol creates a chemiluminescent signal, the intensity of which is attributed to the antigen concentration. The encapsulation of Luminol molecules within the cavities of the ZIF-67 framework protects luminol from premature oxidation, improves its stability and prolongs luminol shelf life. In addition, ZIF-67 is biocompatible and possess a large surface area, which provide a large amount of luminol to be encapsulated, rising the sensitivity of the detection system. On the other hand, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is analytically the most useful oxidant of luminol and It is the end result of numerous cellular oxidation processes, and these processes can be readily connected to optical detection [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Scheme S1B shows the schematic diagram of the experiment of chemiluminescent aptasensor.\u003c/p\u003e \u003cp\u003eSEM images of luminol@ZIF-67 show Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. The images exhibit a uniform rhombic dodecahedral shape, which obviuosly suggest that the presence of luminol did not interfere with the formation of ZIF-67 nanoparticles. The average size of the luminol@ZIF-67 nanoparticles is about 500 nm.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB shows results of size disparity of dynamic light scattering (DLS) of ZIF-67 synthesized and luminol@ZIF-67 that it is further confirmed by the avarages size of nanostructure and size distribution.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC shows the structure information and ZIF-67's crystal phase and luminol@ZIF-67 which studied by XRD analysis. As can be observed, both the ZIF-67 and luminol@ZIF-67 samples have displayed distinctive MOF diffraction peaks due to ptresence of the chractristic peaks in the both XRD patterns at 7.19\u0026deg;, 10.41\u0026deg;, 12.68\u0026deg;, 14.72\u0026deg;, 16.42\u0026deg;, 18.05\u0026deg;, 22.07\u0026deg;, 24.51\u0026deg;, 25.57\u0026deg;, 29.58\u0026deg;, 31.31\u0026deg;, 32.50\u0026deg;, and 43.12\u0026deg; correspondig to (011), (002), (112), (022), (013), (222), (114), (233), (134), (044), (244), (235), and (100) difraction planes that The diffraction peaks closely match the standard specimen card (JCPDS card no. 00-062-1030), suggesting that ZIF-67 has a more complete crystal structure and is more pure [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The positions and intensities of the luminol@ZIF-67 diffraction peaks are found to be comparable to those of ZIF-67. These data corroborate the good crystallinity with luminol@ZIF-67 after luminol loading and demonstrate that ZIF-67 had well integrated within the synthesized composite, which is in line with the SEM results.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe FT-IR spectrum of luminol, ZIF-67, with luminol@ZIF-67 are displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE. The characteristic absorption spectrum of luminol, which have their centers at 1620 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 3400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, are seen in the luminol@ZIF-67 and luminol FT-IR spectra. These bands are attributed to the vibrations that stretch for C\u0026thinsp;=\u0026thinsp;O and N-H as of luminol's primary amine group (-NH2) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. ZIF-67and luminol@ZIF-67 FT-IR spectrum demonstrate the strong bands of absorption at 425 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 990 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 1573 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e that are ascribed to the bending stretching vibrations of Co\u0026ndash;N (metal-ligand), C\u0026ndash;N,andC\u0026thinsp;=\u0026thinsp;N [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. These observation indicate the successful encapsulation of luminol within ZIF-67.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe luminol@ZIF-67 XPS survey spectra are revealed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. As can be seen from the detected C 1s, N 1s, O 1s, and Co 2p peaks, the XPS survey suggests the existence of C,N,O, and Co. Cobalt atoms in Co\u0026ndash;N bonds are represented by the detected peaks on 785.8 eV and 801.2 eV in the high-resolution XPS spectrum of Co2p in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], while Co 2p3/2 and Co 2p1/2 of Co\u003csup\u003e2+\u003c/sup\u003e are represented by the peaks at 781.0 eV and 796.7 eV, respectively, and can be credited to cobalt atoms in Co\u0026ndash;O bonds [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The carbon atom within the \u0026ndash;C\u0026ndash;NH2 group and the \u0026ndash;C\u0026thinsp;=\u0026thinsp;O\u0026ndash; group from luminol are identified by peaks centered in 286.2 eV and 288.3 eV, respectively, in the high-resolution XPS spectrum of C 1s in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The presence of carbon in the C\u0026ndash;N, O\u0026ndash;C\u0026ndash;NH, and \u0026ndash;C\u0026thinsp;=\u0026thinsp;N\u0026ndash; groups from luminol is attributed to the peaks that appear at 399.31 eV, 399.75 eV, and 400.45 eV in the high-resolution XPS spectrum of N 1s in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, respectively. The peak at 398.92 eV is attributed to N\u0026ndash;Co [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. These findings further demonstrate that luminol was successfully encapsulated within ZIF-67.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA depicts the XRD pattern of PVP, NiO and PVP/NiO nanocomposite. XRD pattern of PVP shows two broad peaks aproximately 2θ of 11.5\u0026deg; and 21.2\u0026deg; and these peaks are observed in PVP/NiO at 2θ of 11.6 and 21.5\u0026deg;. It is found that XRD pattern of NiO and PVP/NiO samples samples clearly exhibit the diffraction peaks of (111),(200),(220),(311),and (222) plane, indexed into face-centred-cubic (fcc) structured NiO (JCPDS card no. 00-047-1049) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. XRD patterns of PVP/NiO nanocomposite shows the diffraction peaks that they are low and broad due to the PVP anchoring in nanocomposite.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB depicts the FT-IR spectra of PVP and PVP/NiO nanocomposite. Both spectra show a broad band at about 3445 cm\u003csup\u003e-1\u003c/sup\u003e attributed to O-H group bending vibration and adsorbed water molecules [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. As observed, a band located at 1675 cm\u003csup\u003e-1\u003c/sup\u003e is related to the carbonyl (C\u0026thinsp;=\u0026thinsp;O) stretching vibration of PVP [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Around 2950 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is where the band associated with PVP's CH\u003csub\u003e2\u003c/sub\u003e asymmetric vibration of stretching is located [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and the band at around 1440 cm\u003csup\u003e-1\u003c/sup\u003e is ascribed to the vibration of heterocyclic in PVP [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. A strong peak at 470 cm\u003csup\u003e-1\u003c/sup\u003e in PVP/NiO is attributed to the Ni-O stretching mode [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. These characteristic peaks demonestrate the successful synthesis of PVP/NiO nanocomposite.\u003c/p\u003e \u003cp\u003eThe peroxidase-like activity of luminol@ZIF-67 was demonestrated through the catalytic oxidation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, as exhibited in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. The peak of absorption at 652 nm was used to study the catalytic activity [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Results show that luminol@ZIF-67 reveals a maximum absorbance at 652 nm, asociated with the oxidation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, demonestrating the catalytic activity of Luminol@ZIF-67 is related to presence of luminol. Co-imidazole MOF (ZIF-67) was shown in previous research to be an efficient CL catalyst for the luminol-H2O2 CL reaction [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Several substances have been included to the reaction while maintaining equivalent luminol concentration in order to further elucidate the catalytic activity for luminol@ZIF-67. Imidazole may enhance the catalytic activity on peroxidase-like activity, as seen by the control studies in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, which demonstrate that the catalytic activity within the existence of 2-methylimidazole in solutions is greater than that of luminol alone. Moreover, Michaelis-Menten constant (K\u003csub\u003em\u003c/sub\u003e) was calculated using the Lineweaver-Menten plot [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The relatively small K\u003csub\u003em\u003c/sub\u003e values for H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (0.078 mM) that lower than the Km of horseradish peroxidase [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], nanostructured polymer membrane [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], NiO thin film [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] and NiO nanoparticles [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] demonestrate an increased affinity between H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and luminol@ZIF-67, indicating higher affinity of luminol@ZIF-67 for H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, the catalytic efficiency of ZIF-67 and luminol@ZIF-67 in an alkaline solution was examined. The catalytic efficiency of luminol@ZIF-67 is demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC to be greater than roughly six times that of luminol or ZIF-67 alone in the CL system. This enhancement is asociated with the tendency of luminol to form dimers in aqueous solution, a problem that was mitigated by incorporating luminol into ZIF-67 [\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Therefore, the synergistic catalytic properties of both luminol and ZIF-67 can promote the luminol@ZIF-67 catalytic activity.\u003c/p\u003e \u003cp\u003eTo examine local electron conversion resulted from catalytic Luminol@ZIF-67 activity in the CL reaction by activating H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, phenylhydrazine and thiourea employed to assess the existence of \u003csup\u003e\u0026bull;\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003eand\u003csup\u003e\u0026bull;\u003c/sup\u003eOH radicals [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD displays that the existence of phenylhydrazine into the sensor structure led to decrease the CL signal, revealing that luminol@ZIF-67 enhance the creation of \u003csup\u003e\u0026bull;\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e. Results show that thiourea as scavenger in \u003csup\u003e\u0026bull;\u003c/sup\u003eOH effectively inhibited the CL signal, implying that \u003csup\u003e\u0026bull;\u003c/sup\u003eOH is an essential intermediate in the CL system catalyzed by luminol@ZIF-67.\u003c/p\u003e \u003cp\u003eFurthermore, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE indicates that the CL emitter corresponds to the excited-state 3-aminophthalate anion, as the highest emission wavelength for luminol around 440 nm is the same for luminol@ZIF-67-hemin [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Furthermore, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF compares the CL signal's level in air, N\u003csub\u003e2\u003c/sub\u003e, and O\u003csub\u003e2\u003c/sub\u003e environments, demonstrating the critical role that oxygen plays in our CL aptasensor.\u003c/p\u003e \u003cp\u003eFor optimization the conditions for CL aptasensor the effect of ratio of PVP/NiO-ssDNA to luminol@ZIF-67-apt, antigen incubation time, pH of the buffer, NaOH concentration, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration and flow rate on sensitivity of the proposed aptasensor were investigated, and results show in Figure S2, respectively. Results indicate maximum signal of CL aptasensor is obtained in equal ratio of PVP/NiO-ssDNA to luminol@ZIF-67-apt, 20 minutes for antigen incubation time, buffer solution with pH of 7.4, 0.05 M NaOH of, 0.15 M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, the main pump's flow rate was 25 r/min, respectively. Therefore, these conditions were selected as the optimal aptasensor.\u003c/p\u003e \u003cp\u003eUnder optimized condition, the obtained CL aptamer signal for various concentrations of the target antigen is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA. As seen, there is good linearity with increasing concentrations of the target antigen and resulted signal of aptasensor (PVP/NiO-ssDNA/luminol@ZIF-67-apt). The analytical performance of the aptasensor was assessed by measuring the resulted signal and observed that the signal of aptasensor is strongly correlated with the target antigen concentration (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.99946) with equation: I\u0026thinsp;=\u0026thinsp;6109.2179\u0026thinsp;+\u0026thinsp;431.9149 Log C\u003csub\u003eCYFRA 21\u0026minus;1\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The limit of detection (LOD) reaches as low as 3.7\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e ng/mL based on calculating the 3σ/S criterion (where \u0026lsquo;S\u0026rsquo; is the slope of the calibration plot and σ is the standard deviation of the blank). These observations indicate the simplified detection of lung cancer marker via the generation of CL signal using PVP/NiO-ssDNA/luminol@ZIF-67-apt aptasensor. The clinically relevant serum concentration of CYFRA 21\u0026thinsp;\u0026minus;\u0026thinsp;1 in lung cancer patients typically ranges from 1 to 10 ng/mL, with levels above 3.3 ng/mL considered indicative of malignancy. The aptasensor developed in this study exhibits a wide linear detection range from 10⁻⁵ to 10⁴ ng/mL and an ultralow detection limit of 3.7 \u0026times; 10⁻⁵ ng/mL, allowing for accurate detection of even trace levels of CYFRA 21\u0026thinsp;\u0026minus;\u0026thinsp;1. This confirms its practical applicability for clinical screening and early-stage diagnosis, providing a robust and sensitive method for monitoring lung cancer biomarkers in patient samples. The comparison between current study and with previously reported CYFRA 21\u0026thinsp;\u0026minus;\u0026thinsp;1 sensors is presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. It demonstrates that the present CL aptamer nanoprobe as selective sensor offers notably sensitivity and comparable or better performance compared to some previously reported sensors.\u003c/p\u003e \u003cp\u003e \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\u003eComparision between the current study and with previously reported CYFRA 21\u0026thinsp;\u0026minus;\u0026thinsp;1 sensors.\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\u003eMethod\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReagent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLOD (ng/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLinear range (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\u003eCL aptasensor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePVP/NiO-ssDNA/luminol@ZIF-67-apt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.7\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e to 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis work\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlphaScreen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAlphaLISA kit based on antibody coated on AlphaLISA acceptor beads and donor beads coated with streptavidin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.08 to 500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eECL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etris(2,29-bipyridyl)ruthenium (II) complex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 to 30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eECL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMagnetic beads loaded with conductive carbon black\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.14\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e to 10\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eECL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAntibody/3D graphene @ Au NPs/GCE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.25 to 800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eECL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAntibody/3D graphene/chitosan/glutaraldehyde/GCE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.043\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1 to 150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eECL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAntibody/toluidine blue /AuNPs@MoS\u003csub\u003e2\u003c/sub\u003e@Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5 to 50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eECL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAntibody/3-mercaptopropionic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.08\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e to 10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eECL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMoOx quantum dots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e to 350\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eECL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eToluidine blue/AuNPs/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e-MXene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5 to 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEIS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAntibody/amino terminal groups of PTNH\u003csub\u003e2\u003c/sub\u003e polymer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.7\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e to 0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEIS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAntibody/Au NPs@CMK-3@CMWCNTs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e to 10\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIRA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eenzyme immunoassay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e----\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eREI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eamino-Antibody/Ag/BSA/Antibody/MB/CdTe/MoS\u003csub\u003e2\u003c/sub\u003e/GCE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e to 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUV-Vis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAntibody/AuNPs)/reduced graphene oxide/indium tin oxide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e to 20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLSPR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAntibody/Au nanorods\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.496 to 48.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAu @Ag NRs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.9\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e\u0026thinsp;to 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eCL: Chemiluminescent; ECL: Electrochemiluminescent; EIS: Electrochemical impedance spectroscopy; IRA: Immunoradiomentric assay; REI: Ratiometric Electrochemical immunoassay; UV-Vis: UV-Visible spectrophotometer; LSPR: Localized Surface Plasmon Resonance; SERS: Surface-enhanced Raman scattering\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe consistency of the proposed aptasensor was examined by analyzing five aptasensors fabricated under same conditions and at a CYFRA 21\u0026thinsp;\u0026minus;\u0026thinsp;1 concentration of 1 ng/mL. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea shows that the RSD is found to be 3.71%, indicating the reproducibility of the aptasensor. Another important property attribute of the proposed aptasensor is its long-term stability. It was evaluated during a thirty-three-day period. The results demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB reveal that initial resistances are maintained at 98.22% and 97.87% after 20 and 33 days, respectively, indicating appropriate stability.\u003c/p\u003e \u003cp\u003eThe specificity of the fabricated aptasensor was also evaluated under optimal conditions. The aptasensor\u0026rsquo;s response to potential interferences such as neuron-specific enolase (NSE), carcinoembryonic antigen (CEA), alpha fetal protein (AFP), squamous cell carcinoma antigen (SCC), immunoglobulin G (IgG) and BSA were recorded and juxtaposed with the response of the designed aptasensor. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC shows that the CL signal of the fabricated aptasensor to CYFRA 21\u0026thinsp;\u0026minus;\u0026thinsp;1 remarkably surpassed that of other compounds, highlighting that the aptasensor possesses a distinctive recognition capability towards CYFRA 21\u0026thinsp;\u0026minus;\u0026thinsp;1 and corroborates its acceptable specificity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo examine the practicability of the aptasensor for CYFRA 21\u0026thinsp;\u0026minus;\u0026thinsp;1, recovery tests were conducted which involved the assay of four different concentrations (0.50, 1.00, 10.00 and 20.00 ng/mL of CYFRA 21\u0026thinsp;\u0026minus;\u0026thinsp;1) in a human serum sample, using the standard addition method. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e summarizes the obtained results and illustrates to acceptable of the recoveries in range of 96.00 to 99.30% and RSD value from 3.41\u0026ndash;4.07%. the obtained results of the commercially available ELISA assay are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. As found, the responses of the proposed aptasensor are close those of the ELISA assay, suggesting that the suggested aptasensor can detect CYFRA 21\u0026thinsp;\u0026minus;\u0026thinsp;1 in actual samples with sufficient accuracy and validity. Moreover, it demonstrates a potential for precise clinical applications and pharmaceutical researches.\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\u003eThe result of CYFRA 21-1detection in human serum samples.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\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=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSpiked\u003c/p\u003e \u003cp\u003e(ng/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003ePresent aptasensor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eELISA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRelative error (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003efound (ng/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRecovery (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRSD (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003efound (ng/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRecovery (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRSD (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e96.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e98.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2.04082\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e97.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e98.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.02041\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e99.20\u003c/p\u003e \u003c/td\u003e \u003ctd 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colname=\"c8\"\u003e \u003cp\u003e-0.15136\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eA CL aptasensor based on an aptamer conjugate was created in this study to identify CYFRA 21\u0026thinsp;\u0026minus;\u0026thinsp;1 as a biomarker for lung cancer. This CL aptasensor offers several advantages: (1) The use of luminol@ZIF-67-apt as probe remarkably improved sensitivity in the CL system, (2) luminol@ZIF-67 effectively addresses the issue of luminol dimer formation in solution, thereby dramatically improving the CL signal, and (3) The incorporation of PVP/NiO-ssDNA effectively minimizes background interference during CYFRA 21\u0026thinsp;\u0026minus;\u0026thinsp;1 detection. These benefits demonstrated the sensing platform's promise in intricate clinical settings, as demonstrated by the accurate assessment of CYFRA 21\u0026thinsp;\u0026minus;\u0026thinsp;1 amounts in actual samples. An encouraging substitute of CYFRA 21\u0026thinsp;\u0026minus;\u0026thinsp;1 detection for human serum samples was offered by the developed aptasensor. The aptasensor showed significant performance characteristics, including a low detection limit (3.7\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e ng/mL), broad linear range (10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e to 10\u003csup\u003e4\u003c/sup\u003e ng/mL), remarkable reproducibility, and long-term stability (maintaining initial resistance at 97.87% after 33 days). Moreover, the aptamer strategy improves the selectivity of the aptasensor, reducing the likelihood of false positives and ensuring reliable detection. Future work could focus on further optimizing the aptasensor design and expanding its applications in the field of cancer diagnostics in clinical applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors did not receive support from any organization for the submitted work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWieskopf B, Demangeat C, Purohit A, Stenger R, Gries P, Kreisman H, Quoix E (1995) Cyfra 21-1 as a biologic marker of non-small cell lung cancer. Evaluation of sensitivity, specificity, and prognostic role. Chest 108:163-169.\u003c/li\u003e\n\u003cli\u003eMuraki M, Tohda Y, Iwanaga T, Uejima H, Nagasaka Y, Nakajima S (1996) Assessment of serum CYFRA 21‐1 in lung cancer. 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Bioelectrochemistry 148:108230.\u003c/li\u003e\n\u003cli\u003eMuraki M, Tohda Y, Iwanaga T, Uejima H, Nagasaka Y, Nakajima S (1996) Assessment of serum CYFRA 21-1 in lung cancer. Cancer 77:1274-1277.\u003c/li\u003e\n\u003cli\u003eWang J, Yang X, Hua X, Li Y, Jin B (2023) Novel Ratiometric Electrochemical Biosensor for Determination of Cytokeratin 19 Fragment Antigen 21-1 (Cyfra-21-1) as a Lung Cancer Biomarker. Analytical Letters 56:2708-2724.\u003c/li\u003e\n\u003cli\u003eJoshi S, Guruprasad G, Kulkarni S, Ghosh R (2022) Reduced Graphene Oxide Based Electronic Sensors for Rapid and Label-Free Detection of CEA and CYFRA 21-1. IEEE Sensors Journal 22:1138-1145.\u003c/li\u003e\n\u003cli\u003eChakraborty D, Mukherjee A, Ethiraj KR (2022) Gold nanorod-based multiplex bioanalytical assay for the detection of CYFRA 21-1 and CA-125: towards oral cancer diagnostics. 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Sensors and Actuators Reports 7:100198.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Supplementary Files","content":"\u003cp\u003eScheme S1 and Figure S1 are not available with this version\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Chemiluminescence, Cell lung cancer, Cytokeratin 19 fragment, Cobalt-based metal-organic framework, Polyvinyl Pyrrolidone/Nickel Oxide","lastPublishedDoi":"10.21203/rs.3.rs-6891104/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6891104/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHerein, a chemiluminescent (CL) aptasensor was proposed constructed by aptamer-aptamer bioconjugate for ultrasensitive determination of CYFRA21-1 as a lung cancer biomarker. The sensing mechanism was based on the incorporation of luminol into a cobalt-based metal–organic framework (MOF), also called ZIF-67, to generate a composite of the luminol@ZIF-67. And then, perceptional complex was modified with CYFRA21-1 specific aptamer (luminol@ZIF-67-apt) as signal probe. From this, ssDNA was immobilized on PVP/NiO nanocomposite which had been functionalized, allowing hybridization with the aptamer by complementary base pairing. In the presence of CYFRA 21-1, the target-CYFRA 21-1/luminol@ZIF-67-apt/PVP/NiO-ssDNA sandwich-like ternary complex could be assembled and induced the generation of intense CL emission. Under the optimized conditions, the sensor showed a broad linear detection ranging from 10\u003csup\u003e⁻5\u003c/sup\u003e to 10⁴ ng/mL, with a detection limit (LOD) of 3.7 × 10\u003csup\u003e⁻\u003c/sup\u003e⁵ ng/mL. Reproducibility (RSD = 3.81%) and long-term stabilities was achieved, where the signal was respectively retained to 98.22 and 97.87% over 20 and 33 days. The recovery experiments in spiked human serum samples were in the range from 96.00% to 99.30%, and RSDs were varied from 3.41% to 4.07%, indicating that the aptasensor had good stability and good potential in complex biological matrices. Such a strategy holds a great promise for ultrasensitive detection of protein biomarkers for clinical bio-analysis.\u003c/p\u003e","manuscriptTitle":"An innovative technique for sensitive detection of carcinoembryonic antigen as a cell lung cancer marker","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-04 06:17:11","doi":"10.21203/rs.3.rs-6891104/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-18T08:07:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-16T10:33:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-16T10:31:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microchimica Acta","date":"2025-06-14T00:31:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6e649160-9591-427f-8523-9a64b609f931","owner":[],"postedDate":"July 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-03T16:07:00+00:00","versionOfRecord":{"articleIdentity":"rs-6891104","link":"https://doi.org/10.1007/s00604-025-07590-3","journal":{"identity":"microchimica-acta","isVorOnly":false,"title":"Microchimica Acta"},"publishedOn":"2025-10-30 15:56:56","publishedOnDateReadable":"October 30th, 2025"},"versionCreatedAt":"2025-07-04 06:17:11","video":"","vorDoi":"10.1007/s00604-025-07590-3","vorDoiUrl":"https://doi.org/10.1007/s00604-025-07590-3","workflowStages":[]},"version":"v1","identity":"rs-6891104","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6891104","identity":"rs-6891104","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}