CeO2 /g-C3N4 photoactive nanozymes based colorimetric immunoassay for CEA detection

CeO2 /g-C3N4 photoactive nanozymes based colorimetric immunoassay for CEA detection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article CeO2 /g-C3N4 photoactive nanozymes based colorimetric immunoassay for CEA detection Yan Cheng, Xing Hu, Bing Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7027014/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Sep, 2025 Read the published version in Microchimica Acta → Version 1 posted 14 You are reading this latest preprint version Abstract Enzyme-mimicking nanomaterials have gained significant attention for use in colorimetric immunoassays. However, further enhancing their mimetic enzyme activity remains crucial for improving assay sensitivity. In this study, CeO 2 /g-C 3 N 4 nanozymes were synthesized using a simple hydrothermal method. These materials demonstrated enhanced catalytic activity when exposed to visible light. The irradiation facilitates electron transfer within the CeO 2 /g-C 3 N 4 nanomaterials, leading to the generation of reactive oxygen species (ROS). These ROS then promote the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), causing a distinct color change. Based on this, a novel colorimetric immunoassay platform was developed, using the CeO 2 /g-C 3 N 4 nanomaterials as labels. Under visible light, the color development signal was significantly amplified, resulting in improved sensitivity. This approach showed a linear response for the detection of carcinoembryonic antigen (CEA) in the range of 0.5–30 ng/mL, with a limit of detection (LOD) of 0.13 ng/mL. The assay also demonstrated high specificity and excellent reproducibility. Consequently, this method offers a promising strategy for early cancer diagnosis and holds considerable potential for broader clinical applications. Colorimetric Immunoassay CEA CeO2/g-C3N4 Visible light Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Carcinoembryonic antigen (CEA) is a glycoprotein biomarker with significant relevance across various cancers. Typically, physiological CEA levels are around 5 ng/mL. Its primary clinical utility lies in aiding differential diagnosis, monitoring disease progression, and evaluating treatment efficacy for different carcinoma types. CEA is often overexpressed in several malignancies, including gastric, colorectal, breast, and ovarian cancers, solidifying its importance as a key biomarker in human oncology [ 1 – 3 ]. Multiple analytical methods have been developed for detecting CEA in clinical samples, such as enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), surface-enhanced Raman scattering immunoassays, and electrochemical immunoassays [ 4 – 8 ]. However, these techniques often require sophisticated instrumentation, incur significant costs, and involve complex procedures, limiting their practical application, particularly in resource-limited settings. Addressing these limitations necessitates the development of more robust and efficient detection strategies. Colorimetric immunoassays present a viable alternative. They enable visual signal interpretation without the need for expensive equipment, facilitating real-time analysis of tumor markers in point-of-care settings [ 9 , 10 ]. Nanomaterials possess extensive application potential in analytical detection due to their distinct physicochemical properties. In colorimetric immunoassays, their use as enzyme mimics offers a key advantage, substantially enhancing both sensitivity and selectivity [ 11 – 13 ]. Conventional colorimetric immunoassays rely on reactions between enzyme labels and substrates; however, natural enzymes suffer from limited stability and high preparation costs. In contrast, nanomaterial-based enzyme mimics not only provide efficient catalytic activity but also amplify chromogenic signals through their intrinsic optical properties, thereby improving detection sensitivity and stability. Such nanomaterials have been widely employed for detecting diverse biomarkers, including tumor markers, hormones, and pathogens [ 14 – 16 ]. For instance, Lao et al. designed a CuFe/Fe PBA nanozyme exhibiting exceptional peroxidase (POD)-like activity and developed a colorimetric immunoassay for human epidermal growth factor receptor 2 detection with high sensitive and selective [ 17 ]. Despite advancements in the sensitivity of nanomaterial-based colorimetric immunoassays, persistent challenges remain in multiplex target detection, achieving in situ real-time monitoring, and enhancing the signal-to-noise ratio. Photoreactive nanozymes, encompassing photoluminescent nanomaterials, photothermal effect nanomaterials, and photocatalytically active nanomaterials, present novel strategies to address these limitations by incorporating photo-responsive mechanisms [ 18 – 20 ]. Specifically, photocatalytically active nanomaterials, such as titanium dioxide, and zinc oxide, generate reactive oxygen species (ROS) upon light excitation [ 21 – 23 ]. These ROS drive specific chemical reactions, enabling the integration of optical detection with catalytic amplification and significantly enhancing immunoassay performance. For instance, graphitic carbon nitride (g-C 3 N 4 ) demonstrates exceptional photocatalytic properties with a large specific surface area and suitable band gap [ 24 ]. Furthermore, various heterogeneous catalysts, including ZnIn 2 S 4 /Au/C 3 N 4 , g-C 3 N 4 @MoS 2 , g-C 3 N 4 /CdS, and C 3 N 4 /BiVO 4 , have been extensively explored and successfully applied in biosensor [ 25 – 28 ]. In this study, CeO 2 /g-C 3 N 4 composite photocatalytic nanomaterials were successfully synthesized via a hydrothermal method. These materials were then utilized for constructing a colorimetric immunoassay. Under visible light irradiation, the CeO 2 /g-C 3 N 4 composite nanomaterials exhibit photoinduced electron transitions, generating photogenerated holes (h⁺), which catalytically oxidize TMB to form blue oxidized TMB (TMBox). As the CEA concentration increases, the colorimetric signal intensifies, enabling quantitative detection of CEA through solution absorbance measurements. 2. Experimental 2.1. Chemicals Carcinoembryonic antigen (CEA) and anti-CEA mouse monoclonal antibody (Ab) were purchased from Sangon Biotech Co., Ltd (Shanghai, China). Magnetic beads (-COOH, 100–200 nm, 5 mg mL − 1 ), melamine, cerous nitrate hexahydrate (Ce(NO 3 ) 3 ·6H 2 O), 3,3 '5,5' -tetramethylbenzidine (TMB), N -(3-dimethylaminopropyl)- N ’-ethylcarbodiimide hydrochloride (EDC), and N -hydroxysulfosuccinimide sodium salt (NHS) were purchased from Aladdin Reagent Company (Shanghai, China). Other chemicals used were of analytical reagent grade and required no further purification. 2.2. Preparation of CeO 2 /g-C 3 N 4 nanomaterial The synthesis of carboxylated g-C 3 N 4 nanomaterials followed a method previously reported [ 29 ]. Melamine (5 g) was heated in a muffle furnace at 550°C for 4 h. After cooling, the resulting g-C 3 N 4 was ground into a powder. 1g of this powder was refluxed with 100 mL of 5 M nitric acid at 125°C for 24 h to achieve carboxylation. The product was washed repeatedly with deionized water, centrifuged, and dried overnight at 60°C in a vacuum oven. Furthermore, the CeO 2 /g-C 3 N 4 nanocomposite was prepared via a hydrothermal method. Specifically, 1 g of carboxylated g-C 3 N 4 was dispersed in 60 mL of a mixed solvent containing deionized water and ethanol (1:1 v/v). This dispersion was subjected to ultrasonic treatment for 1 h to achieve homogeneity. Subsequently, 0.434 g of cerium nitrate hexahydrate was introduced into the mixture, followed by vigorous stirring for 30 min. This yielded a homogeneous suspension, which was then transferred to a Teflon-lined stainless-steel autoclave. The autoclave underwent thermal treatment at 180°C for 12 h under forced-air convection. The resulting solid was collected by centrifugation after natural cooling. Soluble impurities were removed through repeated washing cycles involving deionized water and absolute ethanol. Finally, the purified nanomaterial was dried overnight in a vacuum oven at 60°C, yielding the CeO 2 /g-C 3 N 4 nanocomposite. 2.3. Preparation of bioconjugates The bioconjugates of nanomaterial and anti-CEA mouse monoclonal antibody were prepared via the carbodiimide method. Initially, 0.0383 g of EDC and 0.023 g of NHS were dissolved in 2 mL of magnetic bead (MB) or CeO 2 /g-C 3 N 4 solution (1 mg/mL), followed by activation through agitation at RT for 30 min. Then, 200 µL of antibody (Ab) was introduced into the solution, and the mixture was agitated at 4°C overnight. Upon reaction completion, the bioconjugates were isolated through centrifugation and washing procedures, designated as MB-Ab and CeO 2 /g-C 3 N 4 -Ab. The bioconjugates were stored at 4°C for subsequent applications. 2.4. Colorimetric immunoassay for CEA A colorimetric immunoassay for carcinoembryonic antigen (CEA) detection was developed based on a sandwich structure, with the construction principle illustrated in Scheme 1 . Initially, 100 µL of the prepared MB-Ab probe was combined with 100 µL of CEA at varying concentrations and incubated with agitation at RT (25.0 ± 0.5°C) for 35 min, allowing the formation of the MB-Ab@CEA complex. Subsequently, 100 µL of CeO₂/g-C₃N₄-Ab bioconjugates solution was introduced to the mixture. Following an additional 35 min incubation period with agitation at room temperature, the final MB-Ab@CEA@CeO₂/g-C₃N₄-Ab complex was obtained. Magnetic separation was repeated to isolate the immunocomplex, which was then resuspended in 500 µL of acetate-buffered saline (ABS, pH 4.0). The addition of 5 mM TMB solution preceded visible light irradiation (λ ≥ 420 nm) for 4 min to initiate chromogenic development. The resulting solution was analyzed using UV-vis absorption spectroscopy across the 350–700 nm wavelength range. Absorbance values at 370 nm and 650 nm were recorded as analytical measurements. All experimental procedures were conducted at 25.0 ± 0.5°C and performed in triplicate. Preferred position of Scheme 1 3. Results and discussion 3.1 Characterizations of CeO 2 /g-C 3 N 4 nanomaterial Initially, transmission electron microscopy (TEM) was employed to characterize the morphological features of g-C 3 N 4 and CeO 2 /g-C 3 N 4 nanomaterials. As illustrated in Fig. 1 A, g-C 3 N 4 exhibits a sheet-like morphology. CeO 2 nanomaterials with uniform spherical morphology and an average particle size of approximately 50 nm are observed to be dispersed on the g-C 3 N 4 nanosheets (Fig. 1 B). Subsequently, X-ray powder diffraction (XRD) analysis was conducted on CeO 2 , g-C 3 N 4 , and CeO 2 /g-C 3 N 4 respectively. Figure 1 C (curve 'a') reveals a characteristic peak at 2θ = 27.56°, corresponding to the (002) crystal plane of g-C 3 N 4 . The characteristic peaks observed at 2θ = 28.55°, 33.1°, 47.5°, 56.3°, 58.95°, 69.3°, 76.6°, and 79° (curve 'b') correspond to the (111), (200), (220), (311), (222), (400), (331), and (420) crystal planes of CeO 2 , respectively. The presence of these characteristic peaks within the CeO 2 /g-C 3 N 4 composite spectrum (curve 'c') confirms the successful synthesis of the composite material. The carboxyl groups present on the g-C 3 N 4 surface were functionalized as ionic groups, thereby providing specific binding sites for antibodies. As demonstrated in Fig. 1 D, the CeO 2 /g-C 3 N 4 nanomaterials exhibit characteristic absorption bands at 3157 cm − 1 , attributed to the stretching vibration of the C = O bond within the carboxyl functional group. Preferred position of Fig. 1 3.2. The affectation of visible light Under visible light exposure, the TMB solution containing CeO 2 /g-C 3 N 4 nanocomposites exhibits a distinct colorimetric reaction (Fig. 2 A). The initially colorless solution undergoes a transition to blue, accompanied by the emergence of characteristic absorption peaks at 370 nm and 650 nm (curve 'c'). Comparative colorimetric systems employing g-C 3 N 4 , CeO 2 , and CeO 2 /g-C 3 N 4 nanocomposites were constructed. Under identical conditions, TMB oxidation occurred in all three systems, evidenced by the appearance of characteristic absorption peaks at 370 nm and 650 nm. However, the colorimetric reaction within the CeO 2 /g-C 3 N 4 nanocomposite system proved more pronounced, yielding a higher TMBox characteristic peak absorbance compared to the other two systems. To elucidate this phenomenon, ultraviolet-visible diffuse reflectance spectroscopy was performed. As depicted in Fig. 2 B, the bandgap energies were determined as 2.86 eV for CeO 2 , 2.75 eV for g-C 3 N 4 , and 2.55 eV for the CeO 2 /g-C 3 N 4 composite. Consequently, the CeO 2 /g-C 3 N 4 composite nanomaterial exhibits enhanced excitation efficiency under visible light and superior photocatalytic performance. This enhancement is primarily attributed to the effective separation of photogenerated electron-hole pairs and the suppression of their recombination. The proposed photocatalytic mechanism is illustrated in Fig. 2 C. Literature indicates that active species including hydroxyl radicals (•OH), singlet oxygen ( 1 O 2 ), superoxide anion (O 2 •− ), and photogenerated holes (h + ) are generated under visible light irradiation. To identify the species responsible for catalytic coloration, controlled quenching experiments were conducted utilizing isopropyl alcohol (•OH scavenger), tryptophan ( 1 O 2 scavenger), superoxide dismutase (SOD, O 2 •− scavenger), and potassium iodide (KI, h + scavenger). As shown in Fig. 2 D, a significant reduction in characteristic peak absorbance was observed in the KI-containing solution (curve 'b'), indicating that h + plays a pivotal role in the photocatalytic oxidation of TMB. Preferred position of Fig. 2 3.3. Performance of the colorimetric immunoassay The optimization of experimental conditions was conducted to ensure optimal immunoassay performance, encompassing antigen-antibody incubation time, ABS pH, TMB concentration, and nanocomposite concentration. The results are presented in Fig. 3 A, B, C, and D. Optimal conditions were determined as follows: a 35 min incubation time, ABS buffer pH of 4.0, TMB concentration of 5.0 mM, and a CeO 2 /g-C 3 N 4 nanocomposite concentration of 2.0 mg/mL. Under these optimized conditions, the CeO 2 /g-C 3 N 4 based colorimetric immunoassy was employed for the detection of carcinoembryonic antigen (CEA). As illustrated in Fig. 3 E, the absorbance of the resultant blue solution at 650 nm exhibited a linear increase with CEA concentration. The linear regression equation was determined as y = 0.16 logC (ng/mL) + 0.084 (R² = 0.982, n = 24), with a limit of detection (LOD) of 0.13 ng/mL (Fig. 3 F). Preferred position of Fig. 3 3.4. Specificity, stability and repeatability To assess the practicability of the colorimetric immunosensor, its specificity, stability, and repeatability were rigorously evaluated. Initially, the interference effects of several substances-calcium ions (Ca²⁺, 4 mM), glucose (Glu, 100 mg/mL), potassium ions (K⁺, 2.5 mM), ascorbic acid (AA, 40 µM), and prostate-specific antigen (PSA, 10 ng/mL)-were investigated. Experimental results (Fig. 4 A) demonstrated that only the target analyte, carcinoembryonic antigen (CEA), elicited a substantial absorbance response. Conversely, the absorbance values for all interfering substances closely approximated those of the blank control group. Furthermore, when CEA was analyzed in combination with these interferents (Ca²⁺, Glu, K⁺, AA, PSA), the absorbance response exhibited negligible deviation compared to CEA analysis alone (Fig. 4 B). This confirms the minimal impact of these potential interferents on CEA detection. Subsequently, five replicate colorimetric immunosensors were fabricated under identical conditions for detecting CEA. As illustrated in Fig. 4 C, the coefficient of variation (CV) for these measurements was 2.52%, indicating high precision. Additionally, immunosensors stored at 4°C for four weeks retained 95% of their initial absorbance response (Fig. 4 D), confirming excellent long-term stability and repeatability. Preferred position of Fig. 4 3.5. Analysis of real serum samples Real human serum samples were obtained from the First Hospital of Shanxi Medical University in strict accordance with the guidelines approved by the First Hospital of Shanxi Medical University ethical committee (ID:IIT-2025-047). These samples were analyzed using the proposed colorimetric immunoassay. Statistical comparison of the assay results with clinical outcomes was performed using t-test. The analytical results are summarized in Table 1 . For all samples, the experimental t-values (t exp ) were found to be lower than the critical t-value (t crit = 4.30), demonstrating that the colorimetric sensor functions reliably and effectively for the detection of carcinoembryonic antigen (CEA) in human serum. Table 1 Comparison of the assay results for real serum samples by using the developed colorimetric immunoassay and ELISA method. Sample colorimetric immunoassay (mean ± SD, ng/mL) ELISA (ng/mL) t exp 1 5.127 ± 0.52 5.060 0.222 2 8.471 ± 0.13 8.370 1.320 3 6.700 ± 0.12 6.550 2.233 4 13.429 ± 0.59 13.560 -0.388 5 9.333 ± 0.12 9.260 1.016 6 11.963 ± 0.12 11.830 1.949 7 17.187 ± 0.56 17.420 -0.742 8 9.727 ± 0.45 9.330 1.542 9 5.480 ± 0.52 5.920 -1.478 10 3.637 ± 0.50 3.150 1.689 11 0.993 ± 0.13 1.070 -1.017 12 0.550 ± 0.05 0.570 -0.655 13 0.693 ± 0.04 0.700 -0.256 Preferred position of Table 1 4. Conclusion This study developed a colorimetric immunosensor utilizing CeO 2 /g-C 3 N 4 composite nanomaterials for the high sensitivity detection of carcinoembryonic antigen (CEA). This approach integrates visible-light-driven photocatalytic activity with a colorimetric signal amplification strategy, achieving a low detection limit for CEA while demonstrating excellent linearity, specificity, and stability. Compared to conventional methodologies, this immunochromatographic sensor eliminates the requirement for expensive instrumentation, offers operational simplicity, and maintains low cost, rendering it particularly suitable for large-scale screening applications in resource-limited settings. The findings provide novel insights for the development of efficient and reliable bioanalytical tools. This technology exhibits potential for extension beyond CEA detection to encompass the analysis of diverse tumor biomarkers and biological proteins. Through modification of antigen and antibody specificity, the methodology holds broad applicability in fields including early cancer diagnosis, environmental monitoring, and food safety. Declarations Acknowledgements Natural Science Foundation of Shanxi Province (No. 20210302123142) are gratefully acknowledged. Funding This research was funded by the Natural Science Foundation of Shanxi Province (No. 20210302123142). Author information Authors and Affiliations Department of Nuclear Medicine, First Hospital of Shanxi Medical University and Shanxi Medical University, Taiyuan 030001, China Yan Cheng College of Chemistry and Chemical Engineering, Taiyuan University of Technology, Taiyuan 030024, China Xing Hu, Bing Zhang Contributions Author contributions Yan Cheng: Resources; Investigation; Writing-Review & Editing Xing Hu: Methodology; Investigation; Formal analysis Bing Zhang: Validation; Supervision; Funding acquisition; Writing-Review & Editing Corresponding author Correspondence to Bing Zhang. Ethics declarations Ethical approval Blood samples were collected from First Hospital of Shanxi Medical University ethical committee accordance with the guidelines approved by the First Hospital of Shanxi Medical University ethical committee (ID:IIT-2025-047). Competing interest The authors declare no competing interests Human ethics and consent to participate declarations Not applicable. 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Diam Relat Mater 111:108161 Scheme Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Scheme.jpg Scheme 1. The schematic illustration of colorimetric immunoassay for CEA detection. Cite Share Download PDF Status: Published Journal Publication published 25 Sep, 2025 Read the published version in Microchimica Acta → Version 1 posted Editorial decision: Revision requested 19 Jul, 2025 Reviews received at journal 18 Jul, 2025 Reviews received at journal 16 Jul, 2025 Reviews received at journal 13 Jul, 2025 Reviews received at journal 12 Jul, 2025 Reviewers agreed at journal 08 Jul, 2025 Reviewers agreed at journal 08 Jul, 2025 Reviewers agreed at journal 08 Jul, 2025 Reviewers agreed at journal 08 Jul, 2025 Reviewers agreed at journal 07 Jul, 2025 Reviewers invited by journal 07 Jul, 2025 Editor assigned by journal 04 Jul, 2025 Submission checks completed at journal 04 Jul, 2025 First submitted to journal 02 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7027014","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":482441013,"identity":"761a80f8-112b-4e4b-a5d6-c24a32bb226d","order_by":0,"name":"Yan Cheng","email":"","orcid":"","institution":"First Hospital of Shanxi Medical University and Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Cheng","suffix":""},{"id":482441014,"identity":"bfb06882-7b45-42ab-beef-5f6828e1df05","order_by":1,"name":"Xing Hu","email":"","orcid":"","institution":"Taiyuan University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Xing","middleName":"","lastName":"Hu","suffix":""},{"id":482441015,"identity":"4c453963-faac-4663-a313-918d08577175","order_by":2,"name":"Bing Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYDACCSBmbACSzIyND6BiBkRqYW9uNjgA4ROlBUjwHG+TIEqL/OzmZw+/7rDIk49IbKv+2Ha4DmjdNgmGmjs4tTDOOWZuLHtGotjwRmLbjYNthyUYeI6VSTAce4ZTC7NEgpm0ZJtE4sYZMC0SOWYSjA2HcWphk0j/BtdSANYi/wa/Fh6gmZIfgVrm8xxsY4DYwoNfi4RETpk04xmJxA3sjc0SZ86lS7bxpBVbJBzDrUV+Rvo2yZ876hLnN7M//FBRZs3Pz354440PNbi1gIOAB0iAopGBkQ3oO5BQAl4NQIU/QNY1gJh/CCgdBaNgFIyCEQkAjrRXFJZkl5MAAAAASUVORK5CYII=","orcid":"","institution":"Taiyuan University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Bing","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-07-02 08:23:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7027014/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7027014/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00604-025-07524-z","type":"published","date":"2025-09-25T15:57:20+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86328612,"identity":"5900d9ce-315d-46af-92f1-16d39c13b2d3","added_by":"auto","created_at":"2025-07-09 11:33:56","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":90780,"visible":true,"origin":"","legend":"\u003cp\u003eTEM of\u003cstrong\u003e \u003c/strong\u003e(A) g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e and (B) CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e; (C) XRD of (a) g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4, \u003c/sub\u003e(b) CeO\u003csub\u003e2, \u003c/sub\u003e(c) CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e; and (D) FTIR of (a) CeO\u003csub\u003e2, \u003c/sub\u003e(b) g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4, \u003c/sub\u003e(c) CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7027014/v1/6b037c9eb0b96954d6db31c6.jpg"},{"id":86328608,"identity":"1745af3d-b848-4e0f-97df-668e10f8a1ba","added_by":"auto","created_at":"2025-07-09 11:33:56","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":103467,"visible":true,"origin":"","legend":"\u003cp\u003e(A) UV-vis spectra of (a) TMB+ g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e, (b) TMB + CeO\u003csub\u003e2\u003c/sub\u003e, (c) TMB+ g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e/CeO\u003csub\u003e2\u003c/sub\u003e under visible light irradiation [inset:photographs of solution a, b, c]; (B) DRS of (a) g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e, (b) CeO\u003csub\u003e2\u003c/sub\u003e, (c) CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e; (C) The schematic illustration of oxidation TMB by CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e;\u003csub\u003e \u003c/sub\u003e(D) UV-vis spectrums of (a) TMB + CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e, (b) TMB + CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4 \u003c/sub\u003e+ KI, (c) TMB + CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4 \u003c/sub\u003e+ isopropanol, (d) TMB + CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4 \u003c/sub\u003e+ tryptophan, (e) TMB + CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4 \u003c/sub\u003e+ SOD under visible light illumination.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7027014/v1/b9a5c6af6d37632d4b54fe4a.jpg"},{"id":86328611,"identity":"79d73e38-1e70-40c4-9f6e-c0e759afb2b0","added_by":"auto","created_at":"2025-07-09 11:33:56","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":138990,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of (A) pH of HCl, (B) TMB concentration, (C) concentration of CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e, (D) irradiation time of 808 nm laser; (E) UV-vis spectra of colorimetric immunoassay toward different concentrations of CEA; (F) Calibration plots of the colorimetric immunoassay toward different concentrations of CEA.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7027014/v1/4ad5884ebf863db233803f4e.jpg"},{"id":86328609,"identity":"554ea13d-d79a-49e2-9f5f-391009052914","added_by":"auto","created_at":"2025-07-09 11:33:56","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":98127,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Selectivity, (B) anti-interference, (C) Stability and (D) Repeatabilityand of colorimetricimmunoassay.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7027014/v1/482fdb12bec0677f8b20ef31.jpg"},{"id":92430451,"identity":"cc89164c-9994-4fbd-af34-9d30a9bb7518","added_by":"auto","created_at":"2025-09-29 16:04:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1270718,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7027014/v1/d043ee4d-24b1-4e6e-bc5d-1faff4990df3.pdf"},{"id":86328613,"identity":"2e32c849-3bf1-44de-b63e-531718c2479f","added_by":"auto","created_at":"2025-07-09 11:33:56","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":62867,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1. \u003c/strong\u003eThe schematic illustration of colorimetric immunoassay for CEA detection.\u003c/p\u003e","description":"","filename":"Scheme.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7027014/v1/94097174dce4b44e6addd14e.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"CeO2 /g-C3N4 photoactive nanozymes based colorimetric immunoassay for CEA detection","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCarcinoembryonic antigen (CEA) is a glycoprotein biomarker with significant relevance across various cancers. Typically, physiological CEA levels are around 5 ng/mL. Its primary clinical utility lies in aiding differential diagnosis, monitoring disease progression, and evaluating treatment efficacy for different carcinoma types. CEA is often overexpressed in several malignancies, including gastric, colorectal, breast, and ovarian cancers, solidifying its importance as a key biomarker in human oncology [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e]. Multiple analytical methods have been developed for detecting CEA in clinical samples, such as enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), surface-enhanced Raman scattering immunoassays, and electrochemical immunoassays [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, these techniques often require sophisticated instrumentation, incur significant costs, and involve complex procedures, limiting their practical application, particularly in resource-limited settings. Addressing these limitations necessitates the development of more robust and efficient detection strategies. Colorimetric immunoassays present a viable alternative. They enable visual signal interpretation without the need for expensive equipment, facilitating real-time analysis of tumor markers in point-of-care settings [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eNanomaterials possess extensive application potential in analytical detection due to their distinct physicochemical properties. In colorimetric immunoassays, their use as enzyme mimics offers a key advantage, substantially enhancing both sensitivity and selectivity [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. Conventional colorimetric immunoassays rely on reactions between enzyme labels and substrates; however, natural enzymes suffer from limited stability and high preparation costs. In contrast, nanomaterial-based enzyme mimics not only provide efficient catalytic activity but also amplify chromogenic signals through their intrinsic optical properties, thereby improving detection sensitivity and stability. Such nanomaterials have been widely employed for detecting diverse biomarkers, including tumor markers, hormones, and pathogens [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e]. For instance, Lao et al. designed a CuFe/Fe PBA nanozyme exhibiting exceptional peroxidase (POD)-like activity and developed a colorimetric immunoassay for human epidermal growth factor receptor 2 detection with high sensitive and selective [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eDespite advancements in the sensitivity of nanomaterial-based colorimetric immunoassays, persistent challenges remain in multiplex target detection, achieving in situ real-time monitoring, and enhancing the signal-to-noise ratio. Photoreactive nanozymes, encompassing photoluminescent nanomaterials, photothermal effect nanomaterials, and photocatalytically active nanomaterials, present novel strategies to address these limitations by incorporating photo-responsive mechanisms [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. Specifically, photocatalytically active nanomaterials, such as titanium dioxide, and zinc oxide, generate reactive oxygen species (ROS) upon light excitation [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. These ROS drive specific chemical reactions, enabling the integration of optical detection with catalytic amplification and significantly enhancing immunoassay performance. For instance, graphitic carbon nitride (g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e) demonstrates exceptional photocatalytic properties with a large specific surface area and suitable band gap [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. Furthermore, various heterogeneous catalysts, including ZnIn\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e4\u003c/sub\u003e/Au/C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e, g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e@MoS\u003csub\u003e2\u003c/sub\u003e, g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e/CdS, and C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e/BiVO\u003csub\u003e4\u003c/sub\u003e, have been extensively explored and successfully applied in biosensor [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eIn this study, CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e composite photocatalytic nanomaterials were successfully synthesized via a hydrothermal method. These materials were then utilized for constructing a colorimetric immunoassay. Under visible light irradiation, the CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e composite nanomaterials exhibit photoinduced electron transitions, generating photogenerated holes (h⁺), which catalytically oxidize TMB to form blue oxidized TMB (TMBox). As the CEA concentration increases, the colorimetric signal intensifies, enabling quantitative detection of CEA through solution absorbance measurements.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"2. Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Chemicals\u003c/h2\u003e\u003cp\u003eCarcinoembryonic antigen (CEA) and anti-CEA mouse monoclonal antibody (Ab) were purchased from Sangon Biotech Co., Ltd (Shanghai, China). Magnetic beads (-COOH, 100\u0026ndash;200 nm, 5 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), melamine, cerous nitrate hexahydrate (Ce(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO), 3,3 '5,5' -tetramethylbenzidine (TMB), \u003cem\u003eN\u003c/em\u003e-(3-dimethylaminopropyl)-\u003cem\u003eN\u003c/em\u003e\u0026rsquo;-ethylcarbodiimide hydrochloride (EDC), and \u003cem\u003eN\u003c/em\u003e-hydroxysulfosuccinimide sodium salt (NHS) were purchased from Aladdin Reagent Company (Shanghai, China). Other chemicals used were of analytical reagent grade and required no further purification.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e\u003cem\u003e2.2. Preparation of\u003c/em\u003e CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e \u003cem\u003enanomaterial\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eThe synthesis of carboxylated g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e nanomaterials followed a method previously reported [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Melamine (5 g) was heated in a muffle furnace at 550\u0026deg;C for 4 h. After cooling, the resulting g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e was ground into a powder. 1g of this powder was refluxed with 100 mL of 5 M nitric acid at 125\u0026deg;C for 24 h to achieve carboxylation. The product was washed repeatedly with deionized water, centrifuged, and dried overnight at 60\u0026deg;C in a vacuum oven.\u003c/p\u003e\u003cp\u003eFurthermore, the CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e nanocomposite was prepared via a hydrothermal method. Specifically, 1 g of carboxylated g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e was dispersed in 60 mL of a mixed solvent containing deionized water and ethanol (1:1 v/v). This dispersion was subjected to ultrasonic treatment for 1 h to achieve homogeneity. Subsequently, 0.434 g of cerium nitrate hexahydrate was introduced into the mixture, followed by vigorous stirring for 30 min. This yielded a homogeneous suspension, which was then transferred to a Teflon-lined stainless-steel autoclave. The autoclave underwent thermal treatment at 180\u0026deg;C for 12 h under forced-air convection. The resulting solid was collected by centrifugation after natural cooling. Soluble impurities were removed through repeated washing cycles involving deionized water and absolute ethanol. Finally, the purified nanomaterial was dried overnight in a vacuum oven at 60\u0026deg;C, yielding the CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e nanocomposite.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Preparation of bioconjugates\u003c/h2\u003e\u003cp\u003eThe bioconjugates of nanomaterial and anti-CEA mouse monoclonal antibody were prepared via the carbodiimide method. Initially, 0.0383 g of EDC and 0.023 g of NHS were dissolved in 2 mL of magnetic bead (MB) or CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e solution (1 mg/mL), followed by activation through agitation at RT for 30 min. Then, 200 \u0026micro;L of antibody (Ab) was introduced into the solution, and the mixture was agitated at 4\u0026deg;C overnight. Upon reaction completion, the bioconjugates were isolated through centrifugation and washing procedures, designated as MB-Ab and CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e-Ab. The bioconjugates were stored at 4\u0026deg;C for subsequent applications.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Colorimetric immunoassay for CEA\u003c/h2\u003e\u003cp\u003eA colorimetric immunoassay for carcinoembryonic antigen (CEA) detection was developed based on a sandwich structure, with the construction principle illustrated in Scheme \u003cspan refid=\"Sch2\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Initially, 100 \u0026micro;L of the prepared MB-Ab probe was combined with 100 \u0026micro;L of CEA at varying concentrations and incubated with agitation at RT (25.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C) for 35 min, allowing the formation of the MB-Ab@CEA complex. Subsequently, 100 \u0026micro;L of CeO₂/g-C₃N₄-Ab bioconjugates solution was introduced to the mixture. Following an additional 35 min incubation period with agitation at room temperature, the final MB-Ab@CEA@CeO₂/g-C₃N₄-Ab complex was obtained. Magnetic separation was repeated to isolate the immunocomplex, which was then resuspended in 500 \u0026micro;L of acetate-buffered saline (ABS, pH 4.0). The addition of 5 mM TMB solution preceded visible light irradiation (λ\u0026thinsp;\u0026ge;\u0026thinsp;420 nm) for 4 min to initiate chromogenic development. The resulting solution was analyzed using UV-vis absorption spectroscopy across the 350\u0026ndash;700 nm wavelength range. Absorbance values at 370 nm and 650 nm were recorded as analytical measurements. All experimental procedures were conducted at 25.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C and performed in triplicate.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreferred position of\u003c/b\u003e Scheme \u003cspan refid=\"Sch2\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e\u003cem\u003e3.1 Characterizations of\u003c/em\u003e CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e \u003cem\u003enanomaterial\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eInitially, transmission electron microscopy (TEM) was employed to characterize the morphological features of g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e and CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e nanomaterials. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e exhibits a sheet-like morphology. CeO\u003csub\u003e2\u003c/sub\u003e nanomaterials with uniform spherical morphology and an average particle size of approximately 50 nm are observed to be dispersed on the g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e nanosheets (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Subsequently, X-ray powder diffraction (XRD) analysis was conducted on CeO\u003csub\u003e2\u003c/sub\u003e, g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e, and CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e respectively. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003eC (curve 'a') reveals a characteristic peak at 2θ\u0026thinsp;=\u0026thinsp;27.56\u0026deg;, corresponding to the (002) crystal plane of g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e. The characteristic peaks observed at 2θ\u0026thinsp;=\u0026thinsp;28.55\u0026deg;, 33.1\u0026deg;, 47.5\u0026deg;, 56.3\u0026deg;, 58.95\u0026deg;, 69.3\u0026deg;, 76.6\u0026deg;, and 79\u0026deg; (curve 'b') correspond to the (111), (200), (220), (311), (222), (400), (331), and (420) crystal planes of CeO\u003csub\u003e2\u003c/sub\u003e, respectively. The presence of these characteristic peaks within the CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e composite spectrum (curve 'c') confirms the successful synthesis of the composite material. The carboxyl groups present on the g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e surface were functionalized as ionic groups, thereby providing specific binding sites for antibodies. As demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, the CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e nanomaterials exhibit characteristic absorption bands at 3157 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, attributed to the stretching vibration of the C\u0026thinsp;=\u0026thinsp;O bond within the carboxyl functional group.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreferred position of\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.2. The affectation of visible light\u003c/h2\u003e\u003cp\u003eUnder visible light exposure, the TMB solution containing CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e nanocomposites exhibits a distinct colorimetric reaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The initially colorless solution undergoes a transition to blue, accompanied by the emergence of characteristic absorption peaks at 370 nm and 650 nm (curve 'c'). Comparative colorimetric systems employing g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e, CeO\u003csub\u003e2\u003c/sub\u003e, and CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e nanocomposites were constructed. Under identical conditions, TMB oxidation occurred in all three systems, evidenced by the appearance of characteristic absorption peaks at 370 nm and 650 nm. However, the colorimetric reaction within the CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e nanocomposite system proved more pronounced, yielding a higher TMBox characteristic peak absorbance compared to the other two systems.\u003c/p\u003e\u003cp\u003eTo elucidate this phenomenon, ultraviolet-visible diffuse reflectance spectroscopy was performed. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, the bandgap energies were determined as 2.86 eV for CeO\u003csub\u003e2\u003c/sub\u003e, 2.75 eV for g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e, and 2.55 eV for the CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e composite. Consequently, the CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e composite nanomaterial exhibits enhanced excitation efficiency under visible light and superior photocatalytic performance. This enhancement is primarily attributed to the effective separation of photogenerated electron-hole pairs and the suppression of their recombination. The proposed photocatalytic mechanism is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eC.\u003c/p\u003e\u003cp\u003eLiterature indicates that active species including hydroxyl radicals (\u0026bull;OH), singlet oxygen (\u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e), superoxide anion (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e), and photogenerated holes (h\u003csup\u003e+\u003c/sup\u003e) are generated under visible light irradiation. To identify the species responsible for catalytic coloration, controlled quenching experiments were conducted utilizing isopropyl alcohol (\u0026bull;OH scavenger), tryptophan (\u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e scavenger), superoxide dismutase (SOD, O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003escavenger), and potassium iodide (KI, h\u003csup\u003e+\u003c/sup\u003e scavenger). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, a significant reduction in characteristic peak absorbance was observed in the KI-containing solution (curve 'b'), indicating that h\u003csup\u003e+\u003c/sup\u003e plays a pivotal role in the photocatalytic oxidation of TMB.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreferred position of\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Performance of the colorimetric immunoassay\u003c/h2\u003e\u003cp\u003eThe optimization of experimental conditions was conducted to ensure optimal immunoassay performance, encompassing antigen-antibody incubation time, ABS pH, TMB concentration, and nanocomposite concentration. The results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B, C, and D. Optimal conditions were determined as follows: a 35 min incubation time, ABS buffer pH of 4.0, TMB concentration of 5.0 mM, and a CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e nanocomposite concentration of 2.0 mg/mL. Under these optimized conditions, the CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e based colorimetric immunoassy was employed for the detection of carcinoembryonic antigen (CEA). As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, the absorbance of the resultant blue solution at 650 nm exhibited a linear increase with CEA concentration. The linear regression equation was determined as y\u0026thinsp;=\u0026thinsp;0.16 logC (ng/mL)\u0026thinsp;+\u0026thinsp;0.084 (R\u0026sup2; = 0.982, n\u0026thinsp;=\u0026thinsp;24), with a limit of detection (LOD) of 0.13 ng/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreferred position of\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Specificity, stability and repeatability\u003c/h2\u003e\u003cp\u003eTo assess the practicability of the colorimetric immunosensor, its specificity, stability, and repeatability were rigorously evaluated. Initially, the interference effects of several substances-calcium ions (Ca\u0026sup2;⁺, 4 mM), glucose (Glu, 100 mg/mL), potassium ions (K⁺, 2.5 mM), ascorbic acid (AA, 40 \u0026micro;M), and prostate-specific antigen (PSA, 10 ng/mL)-were investigated. Experimental results (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) demonstrated that only the target analyte, carcinoembryonic antigen (CEA), elicited a substantial absorbance response. Conversely, the absorbance values for all interfering substances closely approximated those of the blank control group. Furthermore, when CEA was analyzed in combination with these interferents (Ca\u0026sup2;⁺, Glu, K⁺, AA, PSA), the absorbance response exhibited negligible deviation compared to CEA analysis alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). This confirms the minimal impact of these potential interferents on CEA detection.\u003c/p\u003e\u003cp\u003eSubsequently, five replicate colorimetric immunosensors were fabricated under identical conditions for detecting CEA. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, the coefficient of variation (CV) for these measurements was 2.52%, indicating high precision. Additionally, immunosensors stored at 4\u0026deg;C for four weeks retained 95% of their initial absorbance response (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), confirming excellent long-term stability and repeatability.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreferred position of\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Analysis of real serum samples\u003c/h2\u003e\u003cp\u003e Real human serum samples were obtained from the First Hospital of Shanxi Medical University in strict accordance with the guidelines approved by the First Hospital of Shanxi Medical University ethical committee (ID:IIT-2025-047). These samples were analyzed using the proposed colorimetric immunoassay. Statistical comparison of the assay results with clinical outcomes was performed using t-test. The analytical results are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. For all samples, the experimental t-values (t\u003csub\u003eexp\u003c/sub\u003e) were found to be lower than the critical t-value (t\u003csub\u003ecrit\u003c/sub\u003e = 4.30), demonstrating that the colorimetric sensor functions reliably and effectively for the detection of carcinoembryonic antigen (CEA) in human serum.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComparison of the assay results for real serum samples by using the developed colorimetric immunoassay and ELISA method.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" 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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ecolorimetric immunoassay\u003c/p\u003e\u003cp\u003e(mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, ng/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eELISA\u003c/p\u003e\u003cp\u003e(ng/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003et\u003csub\u003eexp\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e5.127\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5.060\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.222\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e8.471\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8.370\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.320\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e6.700\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6.550\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.233\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e13.429\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e13.560\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-0.388\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e9.333\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9.260\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.016\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e11.963\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e11.830\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.949\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e17.187\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e17.420\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-0.742\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e9.727\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9.330\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.542\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e5.480\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5.920\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-1.478\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e3.637\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.150\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.689\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.993\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.070\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-1.017\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.550\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.570\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-0.655\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.693\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.700\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-0.256\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreferred position of\u003c/b\u003e Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study developed a colorimetric immunosensor utilizing CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e composite nanomaterials for the high sensitivity detection of carcinoembryonic antigen (CEA). This approach integrates visible-light-driven photocatalytic activity with a colorimetric signal amplification strategy, achieving a low detection limit for CEA while demonstrating excellent linearity, specificity, and stability. Compared to conventional methodologies, this immunochromatographic sensor eliminates the requirement for expensive instrumentation, offers operational simplicity, and maintains low cost, rendering it particularly suitable for large-scale screening applications in resource-limited settings. The findings provide novel insights for the development of efficient and reliable bioanalytical tools. This technology exhibits potential for extension beyond CEA detection to encompass the analysis of diverse tumor biomarkers and biological proteins. Through modification of antigen and antibody specificity, the methodology holds broad applicability in fields including early cancer diagnosis, environmental monitoring, and food safety.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNatural Science Foundation of Shanxi Province (No. 20210302123142) are gratefully acknowledged.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Natural Science Foundation of Shanxi Province (No. 20210302123142).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDepartment of Nuclear Medicine, First Hospital of Shanxi Medical University and Shanxi Medical University, Taiyuan 030001, China\u003c/p\u003e\n\u003cp\u003eYan Cheng\u003c/p\u003e\n\u003cp\u003eCollege of\u0026nbsp;Chemistry and\u0026nbsp;Chemical Engineering, Taiyuan University of Technology, Taiyuan 030024, China\u003c/p\u003e\n\u003cp\u003eXing Hu, Bing Zhang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYan Cheng: Resources; Investigation; Writing-Review \u0026amp; Editing\u003c/p\u003e\n\u003cp\u003eXing Hu: Methodology; Investigation; Formal analysis\u003c/p\u003e\n\u003cp\u003eBing Zhang: Validation; Supervision; Funding acquisition; Writing-Review \u0026amp; Editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Bing Zhang.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood samples were collected from First Hospital of Shanxi Medical University ethical committee accordance with the guidelines approved by the First Hospital of Shanxi Medical University ethical committee (ID:IIT-2025-047).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman ethics and consent to participate declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNiedzielska J, Jastrzebski T (2025) Carcinoembryonic antigen (CEA): origin, role in oncology, and concentrations in serum and peritoneal fluid. 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Diam Relat Mater 111:108161\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme ","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\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":"Colorimetric, Immunoassay, CEA, CeO2/g-C3N4, Visible light","lastPublishedDoi":"10.21203/rs.3.rs-7027014/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7027014/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEnzyme-mimicking nanomaterials have gained significant attention for use in colorimetric immunoassays. However, further enhancing their mimetic enzyme activity remains crucial for improving assay sensitivity. In this study, CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e nanozymes were synthesized using a simple hydrothermal method. These materials demonstrated enhanced catalytic activity when exposed to visible light. The irradiation facilitates electron transfer within the CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e nanomaterials, leading to the generation of reactive oxygen species (ROS). These ROS then promote the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), causing a distinct color change. Based on this, a novel colorimetric immunoassay platform was developed, using the CeO\u003csub\u003e2\u003c/sub\u003e/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e nanomaterials as labels. Under visible light, the color development signal was significantly amplified, resulting in improved sensitivity. This approach showed a linear response for the detection of carcinoembryonic antigen (CEA) in the range of 0.5\u0026ndash;30 ng/mL, with a limit of detection (LOD) of 0.13 ng/mL. The assay also demonstrated high specificity and excellent reproducibility. Consequently, this method offers a promising strategy for early cancer diagnosis and holds considerable potential for broader clinical applications.\u003c/p\u003e","manuscriptTitle":"CeO2 /g-C3N4 photoactive nanozymes based colorimetric immunoassay for CEA detection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-09 11:33:46","doi":"10.21203/rs.3.rs-7027014/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-20T00:50:41+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-18T07:52:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-16T07:24:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-13T14:42:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-13T03:15:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"307135882844063520975022402051159486710","date":"2025-07-08T13:04:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"67870218900372350192052375118338496429","date":"2025-07-08T12:35:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"212094483267473634531223391212689209368","date":"2025-07-08T09:59:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"213958989658086280555331638666497944631","date":"2025-07-08T08:25:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"130920626196316097029161647683318182001","date":"2025-07-08T01:26:24+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-07T23:08:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-04T12:03:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-04T10:40:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microchimica Acta","date":"2025-07-02T08:22:00+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":"a9f6e136-9f80-4c14-a74a-1be888e7a7a8","owner":[],"postedDate":"July 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-29T15:59:57+00:00","versionOfRecord":{"articleIdentity":"rs-7027014","link":"https://doi.org/10.1007/s00604-025-07524-z","journal":{"identity":"microchimica-acta","isVorOnly":false,"title":"Microchimica Acta"},"publishedOn":"2025-09-25 15:57:20","publishedOnDateReadable":"September 25th, 2025"},"versionCreatedAt":"2025-07-09 11:33:46","video":"","vorDoi":"10.1007/s00604-025-07524-z","vorDoiUrl":"https://doi.org/10.1007/s00604-025-07524-z","workflowStages":[]},"version":"v1","identity":"rs-7027014","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7027014","identity":"rs-7027014","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}