An electrochemiluminescence immunosensor based on reverse-regulation double-resonance energy transfer and a preparation method and application thereof

Abstract

This invention belongs to the field of biosensing and detection technology, and discloses an electrochemiluminescent immunosensor based on reverse-regulated dual-resonance energy transfer, its preparation method, and its application. Preparation method: The substrate electrode is polished; a p-COFs suspension is drop-coated onto the substrate electrode surface; an EDC / NHS mixed solution is drop-added onto the substrate electrode surface; an Ab1 diluent is drop-added onto the substrate electrode surface; a bovine serum albumin solution is drop-added onto the substrate electrode surface; a CYFRA21-1 antigen solution is drop-added onto the substrate electrode surface; and an Ab2-Co3O4@Co-Fe suspension is drop-added onto the substrate electrode surface to obtain the target sensor. Application of an electrochemiluminescent immunosensor based on reverse-regulated dual-resonance energy transfer in the quantitative detection of CYFRA21-1 in human serum. The sensor of this invention possesses excellent selectivity, stability, and repeatability; it can be directly applied to the detection of human serum samples.

Description

Technical Field

[0001] This invention belongs to the field of biosensing and detection technology, specifically relating to an electrochemiluminescent immunosensor based on reverse-regulated dual-resonance energy transfer, its preparation method, and its application. Background Technology

[0002] Cytokeratin 19 fragment (CYFRA21-1) is a core serum biomarker for epithelial malignancies (non-small cell lung cancer, bladder cancer, etc.), and its trace-level ultrasensitive quantitative detection has important clinical value for early tumor screening, efficacy monitoring and prognostic assessment.

[0003] Traditional detection methods such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), lateral flow immunochromatography, and fluorescence immunoassay, while specific and reliable, generally suffer from insufficient sensitivity, narrow linear range, and weak resistance to interference from complex biological matrices, making it difficult to meet the clinical demand for precise quantification of biomarkers at extremely low concentrations.

[0004] Electrochemiluminescence (ECL) detection technology has become the mainstream technology for biomarker detection due to its advantages such as high sensitivity, low background signal, wide linear range, and strong selectivity. The luminescent material is the core of an ECL sensor, and porphyrin compounds are preferred for high-performance luminescent materials due to their rigid large π-conjugated structure, excellent excited-state performance, and structural tunability. However, monomeric porphyrins are prone to aggregation-induced quenching (ACQ), leading to a significant decrease in luminescence efficiency and limiting their application in highly sensitive ECL systems.

[0005] Covalent organic frameworks (COFs) possess characteristics such as high crystallinity, large specific surface area, structural stability, and covalent bonding. They can suppress ACQ (acoustic emission response) through the spatial confinement effect of the framework, thereby improving luminescence stability and intensity. However, the signal variation amplitude of luminescence enhancement within a single framework is limited, and detection sensitivity remains a bottleneck. Summary of the Invention

[0006] To address the problems of low sensitivity, narrow linear range, easy aggregation and quenching of luminescent material, and limited signal modulation in existing CYFRA21-1 detection technologies, the present invention aims to provide an electrochemiluminescence immunosensor based on reverse-modulated dual-resonance energy transfer, its preparation method, and its application. The sensor of the present invention can achieve ultrasensitive, wide linear range, high selectivity, and high stability quantitative detection of CYFRA21-1.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for fabricating an electrochemiluminescence immunosensor based on reverse-regulated dual-resonance energy transfer, comprising the following steps: (1) Polish the base electrode with a diameter of 4~6mm, rinse it clean and dry it; (2) Drop 8~12μL of p-COFs suspension onto the surface of the substrate electrode obtained in step (1), incubate at 25~37℃ for 2~4h, rinse clean and dry; the p-COFs are porphyrin-based covalent organic framework materials, and the p-COFs suspension is obtained by dispersing p-COFs in PBS buffer at pH=7~8, with a concentration of 2~4mg / mL; (3) Add 4~8 μL of EDC / NHS mixed solution to the surface of the substrate electrode obtained in step (2) and activate it for 30~60 min; the EDC / NHS mixed solution is obtained by dissolving EDC and NHS in PBS buffer at pH=7~8 with a molar ratio of EDC:NHS=1:(1~4) and the concentration of EDC in the EDC / NHS mixed solution is 20~40 mmol / L; (4) Add 6~8μL of Ab1 diluent to the surface of the substrate electrode obtained in step (3), incubate at room temperature for 2~4h, rinse clean and dry; Ab1 is a primary antibody against CYFRA21-1, and the Ab1 diluent is obtained by diluting Ab1 with an antibody diluent that is compatible with it, and the concentration of the Ab1 diluent is 10~20μg / L. (5) Add 3-6 μL of bovine serum albumin solution to the surface of the substrate electrode obtained in step (4), seal the electrode surface for 30-60 min, rinse thoroughly and dry; the bovine serum albumin solution is obtained by dissolving bovine serum albumin in PBS buffer at pH 7-8, and the concentration of the bovine serum albumin solution is 0.1-0.3 wt%. (6) Add 6~8 μL of CYFRA21-1 antigen solution to the surface of the substrate electrode obtained in step (5), incubate at room temperature for 2~4 h, rinse clean and dry; the CYFRA21-1 antigen solution is obtained by dissolving CYFRA21-1 in PBS buffer at pH=7~8, and the concentration of CYFRA21-1 antigen solution is 10 fg / mL~500 ng / mL; (7) 6~8 μL of Ab2-Co3O4@Co-Fe suspension was dropped onto the surface of the substrate electrode obtained in step (5), incubated at room temperature for 2~4 h, rinsed clean and dried to obtain the target electrochemiluminescent immunosensor; Ab2 is a secondary antibody adapted to Ab1, and the Ab2-Co3O4@Co-Fe suspension was obtained by dispersing the Ab2-Co3O4@Co-Fe oxide double-shell nanocage composite material in PBS buffer at pH=7~8, and the concentration of Ab2-Co3O4@Co-Fe suspension was 1~3 mg / mL.

[0008] Preferably, Ab1 is an anti-CYFRA21-1 mouse monoclonal antibody, and Ab2 is a goat anti-mouse IgG antibody.

[0009] Preferably, the substrate electrode is a glassy carbon electrode.

[0010] Preferably, the p-COFs are porphyrin-based covalent organic framework materials prepared based on TAPP and ETBA.

[0011] Preferably, the Ab2-Co3O4@Co-Fe oxide double-shell nanocage composite material is prepared according to the following steps: (i) Mix Co3O4@Co-Fe oxide double-shell nanocages, silane coupling agent, and anhydrous ethanol, and stir at 70~90℃ for 1.5~3h. After the reaction is completed, centrifuge and dry to obtain aminated Co3O4@Co-Fe oxide double-shell nanocages; wherein, the ratio of raw materials is Co3O4@Co-Fe oxide double-shell nanocages: silane coupling agent: anhydrous ethanol = (0.1~0.3) g: (0.15~0.45) mL: (10~25) mL; (ii) Aminated Co3O4@Co-Fe oxide double-shell nanocages were dispersed in PBS buffer at pH 7-8 to obtain a dispersion with a concentration of 1-3 mg / mL. (iii) Dilute Ab2 with a compatible antibody diluent to obtain an Ab2 diluent with a concentration of 1~3 μg / mL; (iv) The dispersion obtained in step (ii) and the Ab2 dilution obtained in step (iii) are mixed at a volume ratio of (2~4) mL: (100~300) μL and covalently coupled at room temperature for 2~4 h. After the reaction is completed, the mixture is dried to obtain the Ab2-Co3O4@Co-Fe oxide double-shell nanocage composite material.

[0012] Preferably, the silane coupling agent is 3-aminopropyltriethoxysilane.

[0013] An electrochemiluminescent immunosensor based on reverse-regulated dual-resonance energy transfer, prepared using the aforementioned method.

[0014] Application of the electrochemiluminescent immunosensor based on reverse-regulated dual-resonance energy transfer in the quantitative detection of CYFRA21-1 in human serum.

[0015] In this invention, porphyrin-based covalent organic framework materials p-COFs and Co3O4@Co-Fe oxide double-shell nanocages can be prepared using existing technologies. For example, p-COFs can be referenced, but not limited to, the literature: Wang Li, Liang Jianing, Fu Kaiqi, Sun Lin, Dong Yan, Synthesis and photodynamic therapy of porphyrin-based covalent organic framework nanoparticles, Journal of Zunyi Medical University, 2024, 47(8), 775~782; Co3O4@Co-Fe oxide double-shell nanocages can be referenced, but not limited to, the literature: Q. Chen, et al. MOF-derived Co3O4@Co-Fe oxide double-shell nanocages as multi-functional specific peroxidase-like nanozyme catalysts for chemo / biosensing and dye degradation. Chemical Engineering Journal 395(2020)125130. The system of this invention exhibits a dual reverse resonance energy transfer effect. The enhanced RET is an intraframe resonance energy transfer that occurs between monomer units within p-COFs, significantly enhancing the intrinsic ECL luminescence signal of the material. The quenching RET is an interfacial resonance energy transfer that occurs from p-COFs to Co3O4@Co-Fe, achieving specific quenching of the ECL signal. Through the reverse synergistic regulation of the dual RETs, the signal response window is effectively broadened, enabling ultrasensitive detection of the target antigen.

[0016] Beneficial Effects: The electrochemiluminescence immunosensor constructed in this invention employs a dual RET reverse synergistic regulation strategy. Through the inverse interaction of in-frame signal enhancement and interface signal quenching, it significantly improves the signal variation amplitude, exhibiting significantly higher sensitivity than traditional single RET systems. The spatial confinement effect of the porphyrin-based covalent organic framework material effectively suppresses aggregation-induced quenching (ACQ), further amplifying the luminescence signal in conjunction with in-frame RET. The Co3O4@Co-Fe oxide double-shell nanocage structure effectively shortens the energy transfer path, resulting in high quenching efficiency and good linearity of concentration response. The sensor exhibits excellent detection performance, with a linear detection range of 10 fg / mL to 500 ng / mL and a detection limit as low as 3.26 fg / mL, while also demonstrating excellent selectivity, stability, and repeatability. It can be directly applied to the detection of human serum samples, with a spiked recovery rate of 95.8% to 101.4% and a relative standard deviation (RSD) of <3.9%, demonstrating strong clinical applicability and suitability for early clinical diagnosis of tumors. Attached Figure Description

[0017] Figure 1The images show the ECL signal response curves (A) of the sensor of this invention for different concentrations of CYFRA21-1 (10 fg / mL~500 ng / mL) and the calibration curve (B) of the ECL response current versus the concentration of CYFRA21-1.

[0018] Figure 2 The results of the stability (A), selectivity (B), and storage stability tests (C) of the sensor of this invention. Detailed Implementation

[0019] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the described embodiments, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0020] In the following examples and performance tests, the PBS buffer used had a concentration of 0.01 mol / L and a pH of 7.4.

[0021] Example 1

[0022] A method for fabricating an electrochemiluminescence immunosensor based on reverse-regulated dual-resonance energy transfer, comprising the following steps: (1) Polish the glassy carbon electrode with a diameter of 4 mm with 0.5 μm Al2O3 polishing powder, and rinse it repeatedly with ultrapure water to remove impurities and oxide layer on the electrode surface. Dry it and set it aside. (2) 8 μL of p-COFs suspension was dropped onto the surface of the glassy carbon electrode obtained in step (1), and incubated at 37°C for 2 h to fix the p-COFs luminescent layer. Then, excess p-COFs were washed away with PBS buffer, dried, and set aside for later use. The p-COFs suspension was obtained by dispersing p-COFs in PBS buffer, and the concentration of the p-COFs suspension was 2 mg / mL. (3) Add 4 μL of EDC / NHS mixed solution to the surface of the glassy carbon electrode obtained in step (2) and activate it for 30 min to activate the active groups on the electrode surface; The EDC / NHS mixed solution was obtained by dissolving EDC and NHS in PBS buffer at a molar ratio of EDC:NHS = 1:1, and the concentration of EDC in the EDC / NHS mixed solution was 20 mmol / L. (4) Add 6 μL of Ab1 dilution solution to the surface of the glassy carbon electrode obtained in step (3), incubate at room temperature for 2 h to allow Ab1 to bind to the active groups on the electrode surface, then wash away the unbound Ab1 with PBS buffer, dry and set aside. Ab1 is an anti-CYFRA21-1 mouse monoclonal antibody. The Ab1 diluent is obtained by diluting Ab1 with the antibody diluent purchased and used in conjunction with it. The concentration of the Ab1 diluent is 10 μg / L. (5) Add 3 μL of bovine serum albumin solution to the surface of the glassy carbon electrode obtained in step (4) to block the electrode surface for 30 min in order to block the non-specific binding sites of Ab1 on the electrode surface and avoid subsequent non-specific adsorption interference with detection. Then wash away excess bovine serum albumin with PBS buffer, dry and set aside. The bovine serum albumin solution was obtained by dissolving bovine serum albumin in PBS buffer, and the concentration of the bovine serum albumin solution was 0.1 wt%. (6) Add 6 μL of CYFRA21-1 antigen solution to the surface of the glassy carbon electrode obtained in step (5), incubate at room temperature for 2 h to allow the antigen to specifically bind to Ab1, then wash away excess CYFRA21-1 antigen with PBS buffer, dry and set aside. The CYFRA21-1 antigen solution is obtained by dissolving the CYFRA21-1 antigen in PBS buffer, and the concentration of the CYFRA21-1 antigen solution is 10 fg / mL to 500 ng / mL. (7) 8 μL of Ab2-Co3O4@Co-Fe suspension with a concentration of 1 mg / mL was dropped onto the glassy carbon electrode obtained in step (5). The electrode was incubated at room temperature for 4 h to allow Ab2-Co3O4@Co-Fe to specifically bind to the antigen and form a stable GCE / p-COFs / Ab1 / BSA / CYFRA21-1 / Ab2-Co3O4@Co-Fe sandwich sensing interface. Then, the unbound Ab2-Co3O4@Co-Fe was washed away with PBS buffer and dried to obtain the target electrochemiluminescent immunosensor. The Ab2 is a goat anti-mouse IgG antibody. The Ab2-Co3O4@Co-Fe suspension is obtained by dispersing the Ab2-Co3O4@Co-Fe oxide double-shell nanocage composite material in PBS buffer. The concentration of the Ab2-Co3O4@Co-Fe suspension is 1 mg / mL.

[0023] In this embodiment, the p-COFs are prepared according to the following steps: (a) Weigh 16.87 mg of 5,10,15,20-tetra(4-aminophenyl)porphyrin (TAPP) and 11.1 mg of 4,4′,4′′,4′′′-(ethylene-1,1,2,2-tetramethyl)tetrabenzaldehyde (ETBA) into a clean beaker, add 1.0 mL of 1,2-dichlorobenzene, 1.0 mL of n-butanol and 0.25 mL of 6M acetic acid solution in sequence, and sonicate for 15 min until the system is homogeneous to obtain a homogeneous mixture; (b) The mixture obtained in step (a) is transferred into a polytetrafluoroethylene reactor and allowed to stand at room temperature of 25°C for 3 hours. Then, it is placed at 120°C for hydrothermal reaction for 48 hours. After the reaction is completed, it is filtered, and impurities are removed by repeated washing with anhydrous ethanol and acetone. The mixture is then dried under vacuum to obtain p-COFs.

[0024] In this embodiment, the Co3O4@Co-Fe oxide double-shell nanocage was prepared according to the following steps: (I) Dissolve 40 mg of cetyltrimethylammonium bromide (CTAB) and 1.16 g of cobalt nitrate hexahydrate (Co(NO3)2·6H2O) in 100 mL of methanol and stir until completely dissolved; separately dissolve 1.31 g of 2-methylimidazole in 100 mL of methanol and stir until completely dissolved; then mix the two solutions, stir for 5 min, let stand at room temperature for 24 h, centrifuge and wash to remove impurities to obtain ZIF-67; (II) Weigh 44 mg ZIF-67, ultrasonically disperse it in 30 mL of anhydrous ethanol, add 10 mL of 10 mg / mL potassium ferricyanide (K3[Fe(CN)6]) aqueous solution, and stir continuously at room temperature for 3 h. (III) The reaction product was centrifuged and washed clean, placed in an air atmosphere, heated to 350°C at a heating rate of 2°C / min, annealed for 2 hours, and then cooled to obtain Co3O4@Co-Fe oxide double-shell nanocages.

[0025] In this embodiment, the Ab2-Co3O4@Co-Fe oxide double-shell nanocage composite material was prepared according to the following steps: (i) Weigh 0.1g of Co3O4@Co-Fe oxide double-shell nanocage, 0.15mL of 3-aminopropyltriethoxysilane (APTES) and 10mL of anhydrous ethanol and mix them. Stir the mixture at 70℃ for 1.5h. After the reaction is complete, centrifuge and dry to obtain aminated Co3O4@Co-Fe oxide double-shell nanocage. (ii) Disperse 4 mg of aminated Co3O4@Co-Fe oxide double-shell nanocages in 2 mL of PBS buffer; (iii) Dilute Ab2 with the antibody diluent purchased and used with it to obtain an Ab2 diluent with a concentration of 1 μg / mL; (iv) Mix 2 mL of the dispersion obtained in step (ii) with 100 μL of the Ab2 dilution obtained in step (iii) and covalently couple the mixture at room temperature for 2 h. After the reaction is complete, dry the mixture to obtain the Ab2-Co3O4@Co-Fe oxide double-shell nanocage composite material.

[0026] Performance testing of electrochemiluminescence immunosensor

[0027] The electrochemiluminescence immunosensor prepared in Example 1 was used to detect the CYFRA21-1 antigen. The specific detection conditions and performance test results are as follows: (a) CYFRA21-1 antigen detection The concentration of the CYFRA21-1 antigen solution in step (6) of Example 1 was sequentially adjusted to 10fg / mL, 500fg / mL, 10pg / mL, 500pg / mL, 10ng / mL, and 500ng / mL, and the sensor was constructed according to the method of Example 1.

[0028] The MPI-E type electrochemiluminescence analysis system (multifunctional chemiluminescence detector) and a three-electrode system (the working electrode is the sensor constructed in this study, the reference electrode is an Ag / AgCl (saturated KCl) electrode, and the counter electrode is a platinum wire electrode) were used for detection in PBS buffer containing 0.8 mol / L potassium persulfate (K2S2O8). The test parameters were set as follows: scan potential range of -1.6V to 0V, photomultiplier tube voltage of 800V, and amplification level of 4x. After the parameters were set, the detector was started to acquire the ECL response signal and the ECL signal value was continuously recorded to ensure the stability and accuracy of the signal acquisition.

[0029] ECL signal data for CYFRA21-1 antigen solution at various concentrations were recorded synchronously throughout the entire test. After the test, the logarithm of the CYFRA21-1 antigen concentration (lg) was used as the statistical unit. c Using (I) as the x-axis and the corresponding ECL response signal (I) as the y-axis, a standard curve is plotted and linearly fitted to clarify the relevant parameters of the sensor's linear relationship.

[0030] Figure 1 The images show the ECL signal response curves (A) of the sensor of this invention to different concentrations of CYFRA21-1 (10 fg / mL~500 ng / mL) and the calibration curve (B) of the ECL response current versus CYFRA21-1 concentration. Figure 1As can be seen, within the concentration range of 10 fg / mL to 500 ng / mL, the ECL response current exhibits a good linear relationship with the logarithmic concentration of CYFRA21-1 antigen, and the linear regression equation is I = 11771.60 - 769.93lg c (Where I is the ECL response current and c is the CYFRA21-1 antigen concentration), correlation coefficient R 2 =0.9857, indicating that the sensor has reliable quantitative detection performance and can be used for accurate quantitative analysis of CYFRA21-1 antigen.

[0031] Table 1 compares the performance of the electrochemiluminescence sensor of this invention with other CYFRA21-1 detection methods. As shown in Table 1, the sensor of this invention exhibits significant competitive advantages in both detection range and detection limit. Its superior performance mainly stems from the excellent electrochemiluminescence activity, good biocompatibility, and efficient signal amplification effect of the composite material used in the sensor construction. These characteristics work synergistically to effectively improve the overall detection performance of the sensor, ultimately achieving highly sensitive and wide-range detection of the CYFRA21-1 antigen.

[0032]

[0033] (ii) Stability assessment The concentration of the CYFRA21-1 antigen solution in step (6) of Example 1 was adjusted to 500 pg / mL, and the sensor was constructed according to the method of Example 1.

[0034] The MPI-E type electrochemiluminescence analysis system and a three-electrode system (the working electrode is the sensor constructed in this study, the reference electrode is an Ag / AgCl (saturated KCl) electrode, and the counter electrode is a platinum wire electrode) were used for detection in PBS buffer containing 0.8 mol / L potassium persulfate (K2S2O8). The test parameters were set as follows: the scan potential range was -1.6V to 0V, the photomultiplier tube voltage was 800V, and the amplification level was 4 times. After the parameters were set, the detector was started and 11 cyclic scans were performed continuously. The ECL response signal of each scan was acquired and recorded in real time.

[0035] Figure 2 A represents the stability evaluation result of the sensor of this invention. Experimental results show that after 11 consecutive cyclic scans, the ECL signal of the sensor did not show significant attenuation, the signal strength remained stable, and the fluctuation range was within an acceptable range. These experimental results demonstrate that the constructed sensor possesses excellent stability, meeting the requirements of long-term, repeated detection by CYFRA21-1, and providing a reliable guarantee for its application in actual serum sample detection.

[0036] (III) Selective Detection For common interfering antigens that may be present in actual serum samples, such as prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), neuron-specific enolase (NSE), and procalcitonin (PCT), the following selective tests were performed in groups: Group 1 (CYFRA21-1): The concentration of the CYFRA21-1 antigen solution in step (6) of Example 1 was adjusted to 10 pg / mL, and the sensor was constructed according to the method of Example 1; Group 2 (Mix): The concentration of CYFRA21-1 antigen solution in step (6) of Example 1 was adjusted to 10 pg / mL, and interfering antigens PSA, CEA, AFP, NSE and PCT were added at the same time. The concentration of each interfering antigen was 100 ng / mL. The sensor was constructed according to the method of Example 1. Group 3 (PSA): In step (6) of Example 1, “interference antigen PSA solution (concentration 100ng / mL)” was used instead of “CYFRA21-1 antigen solution”, and the sensor was constructed according to the method of Example 1; Group 4 (CEA): In step (6) of Example 1, “interference antigen CEA solution (concentration 100ng / mL)” was used instead of “CYFRA21-1 antigen solution”, and the sensor was constructed according to the method of Example 1; Group 5 (AFP): In step (6) of Example 1, “interference antigen AFP solution (concentration 100ng / mL)” was used instead of “CYFRA21-1 antigen solution”, and the sensor was constructed according to the method of Example 1; Group 6 (NSE): In step (6) of Example 1, “interference antigen NSE solution (concentration 100ng / mL)” was used instead of “CYFRA21-1 antigen solution”, and the sensor was constructed according to the method of Example 1; Group 7 (PCT): In step (6) of Example 1, “interference antigen PCT solution (concentration 100ng / mL)” was used instead of “CYFRA21-1 antigen solution”, and the sensor was constructed according to the method of Example 1; All the interfering antigen solutions in the above groups were prepared using PBS buffer.

[0037] The MPI-E type electrochemiluminescence analysis system and a three-electrode system (the working electrode is the sensor constructed in this study, the reference electrode is an Ag / AgCl (saturated KCl) electrode, and the counter electrode is a platinum wire electrode) were used for detection in PBS buffer containing 0.8 mol / L potassium persulfate (K2S2O8). The test parameters were set as follows: the scan potential range was -1.6V to 0V, the photomultiplier tube voltage was 800V, and the amplification level was 4 times. After the parameters were set, the detector was started, and the ECL response signal was acquired and continuously recorded.

[0038] Figure 2 B represents the test results of the selectivity and anti-interference performance of the sensor of this invention on CYFRA21-1. From... Figure 2 As can be seen in section B: The sensor constructed in this invention exhibits excellent recognition selectivity; under conditions of coexistence of multiple interfering antigens, the ECL signal response changes remain at a low level. Experimental results confirm that the sensor of this invention has good anti-interference ability against common antigenic interfering agents and can achieve specific detection of CYFRA21-1.

[0039] (iv) Storage stability test The concentration of the CYFRA21-1 antigen solution in step (6) of Example 1 was adjusted to 10 pg / mL, and multiple sets of sensors of the same specifications were constructed according to the method of Example 1.

[0040] Multiple sets of sensors of the same specifications were sealed and stored in a refrigerated environment at 4℃. After storage for 0 days (fresh sensor), 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and 7 days, the sensors were removed and placed in PBS buffer containing 0.8 mol / L potassium persulfate (K2S2O8) for 30 min to equilibrate. Then, an MPI-E type electrochemiluminescence analysis system and a three-electrode system (the working electrode was the sensor constructed in this study, the reference electrode was an Ag / AgCl (saturated KCl) electrode, and the counter electrode was a platinum wire electrode) were used for detection in PBS buffer containing 0.8 mol / L potassium persulfate (K2S2O8). The test parameters were set as follows: scan potential range of -1.6V to 0V, photomultiplier tube voltage of 800V, and amplification level of 4x. After the parameters were set, the detector was started and the ECL response signal was recorded. Three parallel experiments were set up for each storage time point, and the average value was used for analysis.

[0041] Figure 2 C represents the storage stability of the sensor of this invention. The results show that the ECL signal intensity of the sensor remained stable throughout 7 days of sealed storage at 4℃. Compared with the ECL signal of a fresh sensor (stored for 0 days), the attenuation was less than 15%, and the relative standard deviation (RSD) of parallel experiments was less than 3%. These results confirm that the sensor constructed in this invention has good storage stability, can be stored for a long time under specified storage conditions without significant impact on detection performance, further improving its feasibility and practicality in real-world applications.

[0042] (v) Detection of CYFRA21-1 in serum samples To evaluate the clinical analytical capabilities of the constructed sensor in real samples and to reveal its value in early tumor diagnosis, a spiked recovery experiment was conducted on human serum samples using the sensor of this invention. The steps are as follows: (1) Before use, serum samples of known concentrations should be systematically pretreated to eliminate potential interference and ensure detection accuracy. The specific pretreatment process is as follows: First, place the collected serum samples in a centrifuge at 4℃ and centrifuge at 8000r / min for 15min to remove precipitated impurities and large molecular protein aggregates in the serum. After centrifugation, aspirate the clear serum liquid from the top layer and transfer it to a sterile centrifuge tube. Add bovine serum albumin (BSA) as a stabilizer. The amount added is 10μL of 10mg / mL BSA solution (solvent is PBS buffer) per 1mL of serum sample. Gently invert the centrifuge tube 5-8 times to mix it thoroughly and let it stand for 10min to protect the activity of CYFRA21-1 antigen and prevent its degradation. If the concentration of CYFRA21-1 antigen in the serum sample to be tested exceeds the detection limit of the sensor, use PBS buffer to perform serial dilution of the serum sample to ensure that the concentration of the diluted serum sample is within the linear detection range of the sensor. (2) By adding different gradients of CYFRA21-1 standard to the serum samples with known concentrations pretreated in step (1), serum samples with different amounts of CYFRA21-1 were obtained. In step (6) of Example 1, “serum samples with different amounts of CYFRA21-1” were used instead of “CYFRA21-1 antigen solution”, and the sensor was constructed according to the method of Example 1. (3) The MPI-E type electrochemiluminescence analysis system and a three-electrode system (the working electrode is the sensor constructed in this project, the reference electrode is the Ag / AgCl (saturated KCl) electrode, and the counter electrode is the platinum wire electrode) were used to detect the ECL response signal in PBS buffer containing 0.8 mol / L potassium persulfate (K2S2O8). The test parameters were set as follows: the scan potential range was -1.6V to 0V, the photomultiplier tube voltage was 800V, and the amplification level was 4 times. After the parameters were set, the detector was started and the ECL response signal was collected and recorded in real time. (4) The quantitative detection of CYFRA21-1 in serum samples is based on the linear regression equation I=11771.60-769.93lgc: Substitute the actual measured ECL response signal into the above equation to deduce the concentration of CYFRA21-1 antigen in serum samples. (5) Calculate its spiked recovery rate and relative standard deviation (RSD).

[0043] The results are shown in Table 2.

[0044]

[0045] As shown in Table 2, the spiked recovery rate of the sensor of the present invention is 95.8%~101.4%, and the RSD is less than 3.9%. The above experimental results indicate that the sensor of the present invention can accurately and reliably detect CYFRA21-1 in serum samples, and has good application prospects and practical value in the field of clinical diagnosis.

[0046] Note that the specific references mentioned in Table 1 of this invention are as follows: [1] Ge, S.; Wang, MX; Zhu, SH; Wu, H.; Li, J.; Liu, DS; Huang, QL; Li, SB; Sun, XJ Hypersensitive detection of CYFRA21-1 by SERS dualantibody sandwich method. Sens. Actuators Rep. 2024, 7, 100198. DOI: 10.1016 / j.snr.2024.100198. [2] Hou, MY; Ma, LL; Yang, HX; Si, FC; Liu, YJ Background-free and signal-amplified upconversion fluorescent biosensing platform for sensitive detection of CYFRA21-1. Talanta 2023, 262, 124659. DOI: 10.1016 / j.talanta.2023.124659. [3] Hu, K.; Cheng, JM; Wang, KB; Zhao, YQ; Liu, YJ; Yang, HX; Zhang, ZQ Sensitive electrochemical immunosensor for CYFRA21-1 detection based on AuNPs@MoS2@Ti3C2Tx composites. Talanta 2022, 238, 122987.DOI: 10.1016 / j.talanta.2021.122987. [4] Zhang, Q.Q.; Fu, Y.M.; Xiao, K.; Du, C.C.; Zhang, X.H.; Chen,J.H. Sensitive dual-mode biosensors for CYFRA21-1 assay based on the dual-signaling electrochemical ratiometric strategy and “On-Off-On” PEC method. Anal. Chem. 2021, 93, 6801-6807. DOI: 10.1021 / acs.analchem.1c00746. [5] Wang, M.L.; Shu, J.N.; Zhang, R.X.; Yang, D.L.; Teng, G.; Cui, H.Chemiluminescent Fe3O4@Nickel–cobalt double hydroxide magnetic core-shellnanomaterial as an analytical interface for label-free CYFRA21-1immunosensing. Anal. Chem. 2024, 96, 18890-18897.DOI: 10.1021 / acs.analchem.4c05038. [6] Guo, L.; Mu, Z.D.; Qing, M.; Zhou, J.; Li, H.Z.; Wang, L.L.;Zhong, M.Y.; Bai, L.J. A novel signal-on electrochemiluminescenceimmunosensor for the detection of NSCLC antigen biomarker based on new co-reaction accelerators. Adv. Healthc. Mater. 2023, 12, 2202287. DOI: 10.1002 / adhm.202202287.

Claims

1. A method for preparing an electrochemiluminescence immunosensor based on reverse-regulation double-resonance energy transfer, characterized in that, The steps are as follows: (1) Polish the base electrode with a diameter of 4~6mm, rinse it clean and dry it; (2) Drop 8~12μL of p-COFs suspension onto the surface of the substrate electrode obtained in step (1), incubate at 25~37℃ for 2~4h, rinse clean and dry; the p-COFs are porphyrin-based covalent organic framework materials, and the p-COFs suspension is obtained by dispersing p-COFs in PBS buffer at pH=7~8, with a concentration of 2~4mg / mL; (3) Add 4~8 μL of EDC / NHS mixed solution to the surface of the substrate electrode obtained in step (2) and activate it for 30~90 min; the EDC / NHS mixed solution is obtained by dissolving EDC and NHS in PBS buffer at pH=7~8 with a molar ratio of EDC:NHS=1:(1~4) and the concentration of EDC in the EDC / NHS mixed solution is 20~40 mmol / L; (4) Add 6~8μL of Ab1 diluent to the surface of the substrate electrode obtained in step (3), incubate at room temperature for 2~4h, rinse clean and dry; Ab1 is a primary antibody against CYFRA21-1, and the Ab1 diluent is obtained by diluting Ab1 with an antibody diluent that is compatible with it, and the concentration of the Ab1 diluent is 10~20μg / L. (5) Add 3-6 μL of bovine serum albumin solution to the surface of the substrate electrode obtained in step (4), seal the electrode surface for 30-60 min, rinse thoroughly and dry; the bovine serum albumin solution is obtained by dissolving bovine serum albumin in PBS buffer at pH 7-8, and the concentration of the bovine serum albumin solution is 0.1-0.3 wt%. (6) Add 6~8 μL of CYFRA21-1 antigen solution to the surface of the substrate electrode obtained in step (5), incubate at room temperature for 2~4 h, rinse clean and dry; the CYFRA21-1 antigen solution is obtained by dissolving CYFRA21-1 in PBS buffer at pH=7~8, and the concentration of CYFRA21-1 antigen solution is 10 fg / mL~500 ng / mL; (7) 6~8 μL of Ab2-Co3O4@Co-Fe suspension was dropped onto the surface of the substrate electrode obtained in step (5), incubated at room temperature for 2~4 h, rinsed clean and dried to obtain the target electrochemiluminescent immunosensor; Ab2 is a secondary antibody adapted to Ab1, and the Ab2-Co3O4@Co-Fe suspension was obtained by dispersing the Ab2-Co3O4@Co-Fe oxide double-shell nanocage composite material in PBS buffer at pH=7~8, and the concentration of Ab2-Co3O4@Co-Fe suspension was 1~3 mg / mL.

2. The preparation method of reverse-regulation-based bi-resonance energy transfer electrochemiluminescence immunosensor according to claim 1, characterized in that: Ab1 is an anti-CYFRA21-1 mouse monoclonal antibody, and Ab2 is a goat anti-mouse IgG antibody.

3. The method for preparing the electrochemiluminescence immunosensor based on reverse-regulated dual-resonance energy transfer as described in claim 1, characterized in that: The substrate electrode is a glassy carbon electrode.

4. The method for fabricating an electrochemiluminescence immunosensor based on reverse-regulated dual-resonance energy transfer as described in claim 1, characterized in that: The p-COFs are porphyrin-based covalent organic framework materials prepared based on TAPP and ETBA.

5. The method for preparing an electrochemiluminescence immunosensor based on reverse-regulated dual-resonance energy transfer as described in claim 1, characterized in that, The Ab2-Co3O4@Co-Fe oxide double-shell nanocage composite material was prepared according to the following steps: (i) Mix Co3O4@Co-Fe oxide double-shell nanocages, silane coupling agent, and anhydrous ethanol, and stir at 70~90℃ for 1.5~3h. After the reaction is completed, centrifuge and dry to obtain aminated Co3O4@Co-Fe oxide double-shell nanocages; wherein, the ratio of raw materials is Co3O4@Co-Fe oxide double-shell nanocages: silane coupling agent: anhydrous ethanol = (0.1~0.3) g: (0.15~0.45) mL: (10~25) mL; (ii) Aminated Co3O4@Co-Fe oxide double-shell nanocages were dispersed in PBS buffer at pH 7-8 to obtain a dispersion with a concentration of 1-3 mg / mL. (iii) Dilute Ab2 with a compatible antibody diluent to obtain an Ab2 diluent with a concentration of 1~3 μg / mL; (iv) The dispersion obtained in step (ii) and the Ab2 dilution obtained in step (iii) are mixed at a volume ratio of (2~4) mL: (100~300) μL and covalently coupled at room temperature for 2~4 h. After the reaction is completed, the mixture is dried to obtain the Ab2-Co3O4@Co-Fe oxide double-shell nanocage composite material.

6. The method for fabricating an electrochemiluminescence immunosensor based on reverse-regulated dual-resonance energy transfer as described in claim 5, characterized in that: The silane coupling agent is 3-aminopropyltriethoxysilane.

7. An electrochemiluminescent immunosensor based on reverse-regulated dual-resonance energy transfer, prepared by the preparation method according to any one of claims 1 to 6.

8. The application of an electrochemiluminescent immunosensor based on reverse-regulated dual-resonance energy transfer as described in claim 7 in the quantitative detection of CYFRA21-1 in human serum.

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