Immunosensor for detecting confinement composite nano-enzyme enhanced electrochemiluminescence of tumor necrosis factors as well as construction method and application of immunosensor

Abstract

The invention discloses a confinement composite nano-enzyme enhanced electrochemiluminescence immunosensor for detecting tumor necrosis factors and a construction method and application of the immunosensor. The immunosensor is a three-electrode system, and liquid to be detected contains luminol; wherein the working electrode is obtained by covalently immobilizing a tumor necrosis factor specific antibody after an SNF epoxy modified electrode is subjected to confinement of Ni (OH) 2-CeO2 composite nano-enzyme and then closing a non-specific site. The Ni (OH) 2-CeO2 composite nano-enzyme is confined in a nano-channel, the Ni (OH) 2-CeO2 composite nano-enzyme and the nano-channel can synergistically accelerate the process of an oxygen reduction reaction, the modification stability of the nano-enzyme is effectively improved, and a formed immune complex causes increase of interface resistance and steric hindrance of a sensing electrode to cause signal reduction; therefore, high-sensitivity detection of the tumor necrosis factor is realized, and the constructed sensor has the advantages of low detection limit, wide detection range and the like, and is very suitable for being widely applied to the fields of auxiliary clinical diagnosis, large-scale screening, health monitoring and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, and particularly relates to a confined composite nanozyme enhanced electrochemiluminescence immunosensor for detecting tumor necrosis factor, a construction method thereof, and an application thereof. Background Art

[0002] Cytokines are a class of small molecule proteins or polypeptides secreted by cells, which participate in biological processes such as immune regulation, inflammatory response, cell differentiation, and signal transduction. Their abnormal expression is closely related to the occurrence and development of various diseases. Precise detection of cytokines can not only deeply reveal the pathological mechanism of diseases, but also be used for early diagnosis and disease course monitoring, serving personalized medicine and drug research and development. For example, tumor necrosis factor-α (TNF-α) is an important pro-inflammatory cytokine that plays a core role in regulating immune responses, mediating inflammation, and apoptosis. Its abnormal expression is closely related to various diseases such as inflammatory diseases, autoimmune diseases, cancers, and infections. Currently, commonly used cytokine detection methods include enzyme-linked immunosorbent assay (ELISA), flow cytometry, Luminex technology multiplex analysis, etc.

[0003] As disclosed in CN114578063A, a method for detecting human tumor necrosis factor TNF-α by ELISA includes the following steps: S1, sample treatment; S2, ELISA detection. Only a small amount of serum sample is required to detect diseases more conveniently. The detection results such as linearity, repeatability, accuracy, and interval precision in its detection scheme all meet the determination of TNF-α in applicable serum samples, and it has the advantages of simple operation, fast detection speed, quantitative detection, and large batch detection, and has good application prospects.

[0004] CN109142334A discloses a kit for detecting tumor necrosis factor by spatial proximity chemiluminescence method and a detection method thereof. The kit includes: an enzyme label, a luminescence label, an adjuvant, a trigger, and a calibrator; wherein, the calibrator includes calibrators with different concentrations of TNF-α antigen and 0.1M phosphate diluent; the enzyme label includes a peroxidase-labeled TNF-α detection antibody and 0.05M phosphate buffer; the luminescence label includes a 9,10-dihydroacridine-labeled TNF-α capture antibody and 0.05M Tris buffer; the adjuvant includes a luminescence adjuvant and citrate buffer. By using the reagent spatial proximity luminescence analysis detection technology, which is a truly homogeneous chemiluminescence technology, without washing, without a carrier, and without a coating process, it has high detection sensitivity and strong specificity, making the detection results more authentic and reliable; at the same time, the reaction time and reaction steps are optimized to make the operation simpler.

[0005] However, the above methods have the disadvantages of cumbersome operation steps, expensive equipment, and high requirements for the technical level of experimental personnel. Therefore, it is of great significance to develop novel, convenient, and highly sensitive cytokine detection methods.

[0006] Electrochemiluminescence (ECL) is an analytical method that combines electrochemistry and chemiluminescence. It has the advantages of low background, high signal-to-noise ratio, excellent spatiotemporal controllability, high analytical sensitivity, easy integration, and miniaturization, and has potential in many fields such as immunoassay, environmental detection, food safety, drug analysis, and public safety. Among many ECL luminescent reagents, luminol has attracted much attention due to its unique properties such as low oxidation potential, low toxicity, high storage stability, low cost, and easy modification. Using dissolved oxygen instead of the traditional coreactant hydrogen peroxide can solve the problems of poor stability and high biological toxicity of hydrogen peroxide. However, the ECL system based on luminol-dissolved oxygen often has better luminescence intensity under alkaline conditions, and strong ECL signals are difficult to observe under near-neutral conditions. Therefore, it is of great significance to improve the luminescence efficiency of the ECL system of the luminol-dissolved oxygen system under near-neutral conditions to construct an electrochemiluminescence sensor more suitable for biosensing.

[0007] Introducing a good oxygen reduction reaction catalyst to accelerate the reduction reaction process of dissolved oxygen and improve the ROS generation efficiency is an effective means to enhance the ECL of the luminol-dissolved oxygen system. Nanozymes with peroxidase-like activity and catalase-like activity can use dissolved oxygen or hydrogen peroxide as substrates and catalyze the production of reactive oxygen species, and are potential excellent catalysts for catalyzing the oxygen reduction reaction process.

[0008] At present, using nanozymes as coreactant promoters to enhance the electrochemiluminescence of luminol is an important method to improve the sensitivity of luminol-based sensors. Summary of the Invention

[0009] Aiming at the problem that the sensitivity of the luminol-based sensor for detecting tumor necrosis factor needs to be improved, the present invention provides a confined composite nanozyme-enhanced electrochemiluminescence immunosensor for detecting tumor necrosis factor. The immunosensor confines a bimetallic nanozyme in a nanochannel, significantly enhancing the ECL signal of luminol.

[0010] To achieve the above object, the technical solution adopted by the present invention is:

[0011] A confined composite nanozyme-enhanced electrochemiluminescence immunosensor for detecting tumor necrosis factor, the immunosensor is a three-electrode system, and the liquid to be detected contains luminol; wherein the working electrode is an SNF epoxy-modified electrode that covalently immobilizes a tumor necrosis factor-specific antibody after confining Ni(OH)2-CeO2 composite nanozyme, and then seals non-specific sites.

[0012] In the present invention, luminol is used as the electrochemiluminescence signal molecule, and dissolved oxygen is used as the coreactant. The composite nanozyme synthesized by SNF confinement is combined to enhance the electrochemiluminescence efficiency of the luminol-dissolved oxygen system. The Ni(OH)2-CeO2 composite nanozyme is confined in the nanochannel. The CeO2 nanozyme has good chemical stability and unique oxygen storage and release ability, while the conversion of Ni elements with different valence states in the Ni(OH)2 nanozyme also has the ability to catalyze the reduction of oxygen. The two form a composite nanozyme that can synergistically accelerate the process of oxygen reduction reaction, effectively improving the modification stability of the nanozyme and enhancing its catalytic performance at the same time. By functionalizing the outer surface of SNF with epoxy groups to covalently immobilize antibodies to form an immunorecognition interface, when tumor necrosis factor in serum is specifically recognized and captured by the antibodies, the immune complex formed causes an increase in the interfacial resistance and steric hindrance of the sensing electrode, resulting in a signal decrease, thereby realizing the highly sensitive detection of tumor necrosis factor. The constructed sensor has the advantages of low detection limit, wide detection range, simple operation and low cost, so it is very suitable for wide applications in the fields of auxiliary clinical diagnosis, large-scale screening and health monitoring.

[0013] Preferably, the preparation of the working electrode includes the steps of:

[0014] Step 1, removing the micelles after the micelle-containing SNF (mesoporous silica nanochannel film) modified electrode is modified with epoxy silane;

[0015] Step 2, electro-depositing the electrode obtained in Step 1 in the Ce precursor solution and the Ni precursor solution in sequence to obtain an electrode with pore-confined Ni(OH)2-CeO2 composite nanozyme;

[0016] Step 3, incubating the electrode obtained in Step 2 in a buffer solution containing tumor necrosis factor antibody, rinsing and then incubating in BSA solution to obtain the working electrode.

[0017] In the present invention, the composite nanozyme is in-situ synthesized and confined by the mesoporous nanochannels of SNF, which can improve the oxygen vacancy distribution of the nanozyme, making it more capable of adsorbing and catalyzing the reduction of dissolved oxygen in the electrolyte solution. At the same time, the spatial confinement can also significantly improve the stability of the nanozyme modification at the electrode interface. In addition, the porous material provides a dispersion space for the composite nanozyme, effectively increasing the reaction active sites of the nanozyme, increasing the collision efficiency of reaction molecules, and greatly improving the luminescence efficiency of the luminol-dissolved oxygen ECL system.

[0018] Preferably, the specific steps of Step 1 include: immersing the micelle-containing SNF modified electrode in a solution containing epoxy group silane, and then immersing it in an ethanol solution containing inorganic acid to remove the micelles.

[0019] Preferably, the epoxy group-containing silane includes any one of γ-methacryloxypropyltrimethoxysilane, 2-(3,4-epoxycyclohexyl)ethyltriethoxysilane, and epoxy group-containing polyethylene glycol silane; the solvent used includes any one or more of ethanol, methanol, acetone, cyclohexane, and acetonitrile; the molar concentration of the solution containing the epoxy group-containing silane is 2-5 mmol / mL;

[0020] The electrode is immersed in the solution containing the epoxy group-containing silane at room temperature for 0.3-3 h;

[0021] The molar concentration of the inorganic acid in the ethanol solution containing the inorganic acid is 0.01-1 mol / mL, and the electrode is stirred in the ethanol solution containing the inorganic acid for 1-30 min.

[0022] The electrode includes any one of indium tin oxide electrode, glassy carbon electrode, screen-printed electrode, gold electrode, fluorine-doped tin oxide electrode, graphite electrode, and carbon fiber electrode;

[0023] The preparation method of the SNF modified electrode includes electrochemically assisted method, solution growth method, two-phase layer growth method, evaporation-induced self-assembly method, π-π interaction-induced method, epitaxial growth method, strong magnetic field method, electric field method, organic solvent-induced self-assembly method, or any combination of one or more of them;

[0024] Specifically, using The method for preparing the SNF modified electrode by the solution growth method includes the steps:

[0025] Sodium hexadecylsulfonate (CTAB) is dissolved in a water-ethanol mixed solvent as a template surfactant. After adding ammonia water to adjust the alkaline environment, tetraethyl orthosilicate (TEOS), an organosiloxane precursor, is introduced to form a precursor solution; the electrode is immersed in this solution to grow a silica nanochannel film (SNF) on its surface. After the growth is completed, the electrode is taken out, rinsed thoroughly with deionized water and dried with nitrogen, and dried and aged overnight to obtain a modified electrode containing a micelle (SM) template; finally, the electrode is immersed in an HCl-ethanol solution and stirred to remove the micelles to obtain a SNF modified electrode with open pores.

[0026] Another method is to prepare the SNF modified electrode based on the EASA method. CTAB is dissolved in an aqueous solution and the pH is adjusted to acidic. After adding TEOS to form a precursor solution, the electrode is inserted into it and a negative voltage is applied. A pH gradient is formed on the electrode surface by water electrolysis to induce the in-situ growth of the silica nanochannel film; after the film growth is completed, the electrode is taken out, rinsed with deionized water and dried with nitrogen, and then dried and aged to obtain a modified electrode containing a micelle template; finally, the micelles are removed by immersion in an HCl-ethanol solution to obtain a SNF modified electrode with open pores.

[0027] Preferably, the Ce precursor solution includes a solution of any one or more of cerium nitrate, cerium sulfate, and cerium chloride, and the solvents used include deionized water, an aqueous sodium chloride solution, an aqueous potassium chloride solution, a phosphate buffer solution, or a Tris-HCl buffer solution;

[0028] The molar concentration of the cerium salt in the solution of the Ce precursor is 0.01 - 0.5 mol / L;

[0029] The Ni precursor solution includes a solution of any one or more of nickel chloride, nickel sulfate, and nickel nitrate, and the solvents used include deionized water, an aqueous sodium chloride solution, an aqueous potassium chloride solution, a phosphate buffer solution, or a Tris-HCl buffer solution;

[0030] The molar concentration of the nickel salt in the solution of the Ni precursor is 0.01 - 0.2 mol / L.

[0031] Preferably, in step 2, the constant potential range for electrodeposition of the electrode in the Ce precursor solution is -1.0 to -0.3 V, the electrodeposition time is 2 to 360 s, and the temperature is room temperature; preferably, the electrodeposition time is 5 to 30 s, and more preferably the deposition time is 15 s.

[0032] The constant potential range for electrodeposition of the electrode in the Ni precursor solution is -0.02 to -0.3 mA / cm 2 , the electrodeposition time is 2 to 120 s, and the temperature is room temperature. Preferably, the electrodeposition time is 2 to 30 s, and more preferably the deposition time is 10 s.

[0033] Preferably, in step 3, the mass concentration of the tumor necrosis factor antibody in the buffer solution containing the tumor necrosis factor antibody is 5 - 30 μg / mL; the solvents used include any one or more of STE buffer solution, TE buffer solution, or phosphate buffer solution; the solute concentration in the buffer solution is 0.005 - 0.5 mol / L, and the pH is 6.5 - 7.5;

[0034] Preferably, in step 3, the incubation conditions in the buffer solution containing the tumor necrosis factor antibody are incubation at 0 - 5 °C for 0.5 - 5 h; the incubation conditions in the BSA solution are incubation at room temperature for 5 - 30 min.

[0035] The present invention also provides a method for constructing the immunological sensor, including the steps:

[0036] Step 1, removing the micelles after modifying the micelle-containing SNF modified electrode with epoxy silane;

[0037] Step 2, sequentially electrodepositing the electrode obtained in step 1 in the Ce precursor solution and the Ni precursor solution to obtain an electrode with pore-confined Ni(OH)2-CeO2 composite nanozyme;

[0038] Step 3: Incubate the electrode obtained in Step 2 in a buffer solution containing tumor necrosis factor antibody, and after rinsing, incubate it in a BSA solution to obtain a working electrode.

[0039] Step 4: Use the working electrode to form a three - electrode system with a reference electrode and a counter electrode, and perform electrochemiluminescence signal detection on the test solution containing luminol.

[0040] The present invention also provides a method for detecting tumor necrosis factor using the above - mentioned immunosensor, including the steps of: incubating the working electrode in the test solution at 0 - 5 °C for 0.5 - 5 h, rinsing and then performing electrochemiluminescence signal detection; the pH of the test solution is 6.5 - 10, and the molar concentration of luminol is 20 - 180 μmol / L.

[0041] The test solution further includes one or more interfering proteins such as bovine serum albumin, alpha - fetoprotein, and carcinoembryonic antigen, and the concentration of each interfering protein is below 10 ng / mL; the solution environment of the test solution is Tris buffer or phosphate buffer.

[0042] The detection principle is as follows: When there is tumor necrosis factor in the serum sample, the tumor necrosis factor antibody specifically binds to the tumor necrosis factor, and an immune complex is formed on the electrode interface, resulting in an increase in the interface resistance and an increase in the steric hindrance effect, which affects the diffusion and mass transfer of luminol and thus reduces the ECL signal of the working electrode. Therefore, according to the degree of decrease in the electrochemiluminescence signal, the electrochemiluminescence detection of tumor necrosis factor can be realized.

[0043] Preferably, the test sample solution includes a standard solution or a serum sample containing tumor necrosis factor.

[0044] The detection limit of the electrochemistry detection method is below 8.0 fg / mL.

[0045] The trigger luminescence potential of the electrochemiluminescence immunosensor is 0.4 - 0.8 V.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] (1) The SNF - confined Ni(OH)2 - CeO2 composite nanozyme of the present invention enhances the ECL signal of the luminol - dissolved oxygen system. The Ni(OH)2 - CeO2 composite nanozyme is in - situ synthesized and confined in the ultra - small and uniform nanochannel array of SNF, which ensures the ultra - small size of the nano - composite enzyme and improves the distribution of its surface oxygen vacancies, thereby enhancing its catalytic activity for the oxygen reduction reaction, generating more reactive oxygen species at the electrode interface, and further sensitizing the ECL signal of the luminol - dissolved oxygen system.

[0048] (2) The SNF-modified electrode of the present invention has an easily modifiable outer surface. After being modified with epoxy groups, it can covalently immobilize antibodies, thereby constructing an immunosensing interface. When the analyte tumor necrosis factor is present in the solution, the antibodies on the immunosensing interface will specifically capture the tumor necrosis factor to form an immune complex. The steric hindrance effect generated by this complex will hinder the diffusion of luminol to the electrode surface, resulting in a significant decrease in the ECL signal. Based on this signal-off mode, highly sensitive ECL detection of tumor necrosis factor in human serum can be achieved. The immunosensor of the present invention is easy to operate during the detection process. It only needs to incubate the immunosensing electrode with the sample to be tested and then directly measure the electrochemical signal, without complex operation procedures and large instruments, significantly simplifying the detection process.

[0049] (3) The present invention is a study on Ni(OH)2-CeO2 composite nanozyme as a coreactant promoter to enhance the electrochemiluminescence of luminol. It enables the generation of a large number of reactive oxygen species under neutral conditions, improving the ECL signal of the luminol-dissolved oxygen system under near-neutral conditions, which is more conducive to the application of ECL detection methods based on luminol luminophores in the biomedical field. Brief Description of the Drawings

[0050] Figure 1 Top-view TEM images (A) and cross-sectional TEM images (B) of SNF in Example 1; SEM image of SNF / ITO electrode (C); top-view TEM image of Ni(OH)2-CeO2@SNF (D); HAADF-STEM image of Ni(OH)2-CeO2@SNF (E); EDS elemental mapping image of Ni(OH)2-CeO2@SNF (F); cross-sectional TEM image of Ni(OH)2-CeO2@SNF (G); lattice structure of Ni(OH)2-CeO2 nanoparticles (H).

[0051] Figure 2 XPS survey spectra of the surfaces of SNF / ITO, CeO2@SNF / ITO, and Ni(OH)2-CeO2@SNF / ITO electrodes in Example 1 (A); high-resolution XPS spectra of the Si 2p (B), Ce 3d (C), Ni 2p (D), and O 1s (E) energy levels on the surface of the Ni(OH)2-CeO2@SNF / ITO electrode.

[0052] Figure 3ECL-potential diagrams (A) and CV curves (B) of SNF / ITO, CeO2@SNF / ITO, and Ni(OH)2-CeO2@SNF / ITO electrodes in 0.01 M PBS (pH = 7.4) containing 100 μM luminol in Example 1, PMT was 800 V, and the scanning rate was 100 mV / s; (B) The upper figure shows the change of the absolute value of the ECL signal and the signal change rate of Ni(OH)2-CeO2@SNF / ITO with the increase of Ni(OH)2 electrodeposition time (CeO2 deposition time was 15 s); (C) The change of the absolute value of the ECL signal and the signal change rate of Ni(OH)2-CeO2@SNF / ITO with the increase of CeO2 electrodeposition time (Ni(OH)2 deposition time was 10 s); (D) ECL signals of ITO, SNF / ITO, and Ni(OH)2-CeO2@SNF / ITO electrodes at different pH values in 0.01 M PBS or Tris-HCl buffer solution containing 100 μM luminol, the scanning rate was 100 mV / s, and PMT was 600 V; (E and F) ECL responses of different electrodes scanned continuously for 12 cycles, the scanning rate was 100 mV / s, and PMT was 800 V; (G) Comparison of ECL intensities of different electrodes in 0.01 M PBS (pH = 7) containing 100 μM luminol.

[0053] Figure 4CV curves (A) and ECL signals (B) of the Ni(OH)2-CeO2@SNF / ITO electrode in Example 1 in 0.01 M PBS (pH = 7.4) containing 100 μM luminol under different atmospheres, scan rate was 100 mV / s, PMT was 700 V; (C) ECL response of the Ni(OH)2-CeO2@SNF / ITO electrode in the luminol-dissolved oxygen system after adding different radical scavengers, I and I0 represent the ECL signals in the presence and absence of radical scavengers, respectively; (D) Electron paramagnetic resonance (EPR) spectra of different electrodes during CV scanning (-1.0 to 0.8 V) in 90% methanol aqueous solution, scan rate was 100 mV / s; ECL-potential diagrams of the SNF / ITO electrode and the Ni(OH)2-CeO2@SNF / ITO electrode in 0.01 M PBS (pH = 7.4) containing 100 μM luminol when the initial scan potential was -0.2 V and the initial scan direction was forward (E) and backward (F), PMT was 800 V, scan rate was 100 mV / s; UV-visible absorption spectra of different electrodes inserted into 0.1 M NaAc / HAc buffer solution (pH 4.0) containing 0.25 mM TMB with (G) or without (H) 1 mM H2O2 after reacting for 20 min, the inset is a photo of the sample after the reaction (ⅰ: ITO; ⅱ: SNF / ITO; ⅲ: CeO2@SNF / ITO; ⅳ: Ni(OH)2@SNF / ITO; ⅴ: Ni(OH)2-CeO2@SNF / ITO).

[0054] Figure 5 CV curves of SNF / ITO (A) and Ni(OH)2-CeO2@SNF / ITO (B) in 0.1 M KCl solution containing 0.5 mM FcMeOH in Example 1 at different scan rates, the inset is the linear regression curve between the CV peak current and the square root of the scan rate. CV curves measured for SNF / ITO and Ni(OH)2-CeO2@SNF / ITO in 0.01 M PBS (pH = 7.4) under air atmosphere (C) or N2 atmosphere (D); CV curves measured for SNF / ITO (E) and Ni(OH)2-CeO2@SNF / ITO (F) in 0.01 M PBS (pH = 7.4) with or without 100 μM luminol under air atmosphere, scan rate was 100 mV / s.

[0055] Figure 6 For Example 1 in 0.1 M KCl solution containing 2.5 mM [Fe(CN)6] 3- / 4-CV curves (A), DPV curves (B), and EIS curves (C) of different electrodes in 0.1 M KCl solution, with a scan rate of 50 mV / s; (D) ECL signals of different electrodes in 0.01 M PBS (pH = 7.4) containing 100 μM luminol; (E) Ab after incubation with 10 μg / mL Ab for different times TNF-α Electrochemiluminescence signals measured for the Ab TNF-α / Ni(OH)2-CeO2@O-SNF / ITO electrode in 0.01 M PBS (pH = 7.4) containing 100 μM luminol, with a PMT of 800 V and a scan rate of 100 mV / s.

[0056] Figure 7 For (A) in Example 1, TNF-α / Ab after incubation with different concentrations of TNF-α TNF-α Electrochemiluminescence signals measured for the Ab TNF-α / Ni(OH)2-CeO2@O-SNF / ITO electrode for the ratio of ECL intensities (I / I0) before (I0) and after (I) the analysis and detection of different species; (E) Ab TNF-α Change in the ratio of ECL intensities before and after detecting 1 ng / mL TNF-α for the Ab Detailed implementation manners

[0057] In order to make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the following examples are used to further elaborate on the present invention. It should be understood that the specific examples described herein are only used to explain the present invention and are not used to limit the present invention. Any modifications or equivalent replacements made by those skilled in the art on the basis of understanding the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention shall be covered within the protection scope of the present invention.

[0058] The raw materials used in the following detailed implementation manners are all purchased from the market.

[0059] Example 1

[0060] A method for detecting tumor necrosis factor in serum samples by an SNF modified electrode using a Ni(OH)₂-CeO₂ composite nanozyme confined in a nanochannel, covalently immobilizing a tumor necrosis factor antibody on the outer surface of the SNF to form a working electrode, and by an electrochemiluminescence method, specifically comprising the steps:

[0061] (1) Preparation of SNF / ITO by solution growth method

[0062] Weigh 0.16 g of CTAB (cetyltrimethylammonium bromide), dissolve it in a mixed solution of 70 mL of H₂O and 30 mL of ethanol, and continuously stir for 5 min to fully dissolve CTAB. Subsequently, add 100 μL of ammonia water (10%) and 80 μL of TEOS, and continue stirring until a clear, transparent, and bubble-free precursor solution is formed. Immerse the ITO (indium tin oxide) electrode completely into the precursor solution, seal it, and place it in a water bath at 60 °C for 24 h. After the reaction, thoroughly rinse the electrode with a large amount of ultrapure water to remove the residual precursor solution, and dry it with nitrogen. Finally, age the electrode at 100 °C for 12 h to obtain an electrode with micelles (SM) in the nanochannel, named SM@SNF / ITO.

[0063] (2) Epoxy group functionalization process of the SNF electrode

[0064] Immerse the SM@SNF / ITO electrode in ethanol (25 mL) containing 2.26 mM GPTMS (γ-glycidoxypropyltrimethoxysilane), react in the dark for 1 h, then take out the electrode and rinse it with ultrapure water to remove the unreacted siloxane reagent, and then dry it with nitrogen. At this time, there are still micelles in the mesoporous silicon nanochannel, denoted as SM@O-SNF / ITO. Place the SM@O-SNF / ITO electrode in a 0.1 M HCl / ethanol solution and stir for 5 min to remove the micelles, thereby obtaining an electrode with an open nanochannel and an outer surface functionalized with epoxy groups, named O-SNF / ITO.

[0065] (3) Confining Ni(OH)₂ and CeO₂ nanozymes in the SNF nanochannel

[0066] First, use the O-SNF / ITO electrode as the working electrode, place it in a 0.2 M Ce(NO₃)₃ solution, and adopt chronoamperometry to achieve the confinement of CeO₂ nanozyme in the SNF nanochannel by continuously applying a constant cathodic voltage of -0.8 V for 15 s. Subsequently, rinse the electrode surface with ultrapure water to remove the residual electrolyte solution, and dry it with nitrogen. The obtained electrode is denoted as CeO₂@SNF / ITO. Next, place this electrode in a 0.1 M Ni(NO₃)₂ solution and adopt a constant current method (-0.1 mA / cm 2)Electrodeposit for 10 seconds to further confine Ni(OH)2 nanozyme within the nanochannels. The electrode that confines both Ni(OH)2 and CeO2 nanozymes is named Ni(OH)2-CeO2@SNF / ITO.

[0067] (4) Fabrication of immunosensor

[0068] Incubate the Ni(OH)2-CeO2@SNF / ITO electrode in a 0.01M PBS (pH = 7.4) solution containing non-specific antibody Ab against tumor necrosis factor (10 μg / mL) at 4 °C for 1.5 h. Subsequently, rinse off the antibody molecules that are not fully bound to the electrode surface with 0.01M PBS (pH = 7.4). Incubate the obtained electrode in a BSA solution (0.5%) at room temperature for 15 min to block non-specific adsorption sites, obtaining the working electrode of the immunosensor (BSA / Ab TNF-α / Ni(OH)2-CeO2@O-SNF / ITO). TNF-α / Ni(OH)2-CeO2@O-SNF / ITO).

[0069] (5) Electrochemiluminescence detection of tumor necrosis factor

[0070] Incubate the immunosensor (BSA / Ab TNF-α / Ni(OH)2-CeO2@O-SNF / ITO) with TNF-α solutions at different concentrations at 4 °C for 1 h to capture TNF-α through the specific interaction between antigen and antibody. After incubation, gently rinse the electrode with 0.01M PBS (pH = 7.4) to remove unbound antigen molecules. Measure the ECL signals of the electrode before and after the binding of TNF-α. The electrolyte solution for ECL testing is a PBS solution (0.01M, pH = 7.4) containing luminol (100 μM). Trigger the ECL process through cyclic voltammetry scanning, with the potential range of CV scanning being -1.0 V to 0.8 V and the scanning rate being 100 mV / s.

[0071] In step 1, the electron microscope of the ITO electrode (SNF / ITO) modified with silica nano-homoporous membrane prepared by solution growth method is as shown in Figure 1 A- Figure 1 C in.

[0072] Figure 1 As shown in the TEM image of the electrode surface in A in, it can be seen that the SNF structure is complete, the film has no cracks, and the mesoporous channels with uniform pore size (about 2 - 3 nm) are arranged in an irregular worm-like shape. Figure 1The TEM image of the electrode cross-section shown in B reveals that SNF has a long-range ordered nanochannel structure, with a uniform film thickness and a pore length of approximately 101 nm. The intact and crack-free surface of SNF, as well as the cross-section of the long-range ordered nanochannel array, indicate that the film has good mechanical stability. Figure 1 The SEM image in C clearly shows the three-layer structure of the SNF / ITO electrode, including the glass layer, ITO layer, and SNF modification layer of the ITO conductive glass, with distinct boundaries between the layers.

[0073] The electrode Ni(OH)2-CeO2@O-SNF / ITO obtained by electrodeposition in Step 3, where the transmission electron microscopy of the in-situ confined composite nanozyme (Ni(OH)2-CeO2@SNF) in the nanochannels of the silica nano-porous membrane is as Figure 1 shown in D- Figure 1 in H.

[0074] Figure 1 Figure D shows the TEM image of the surface of Ni(OH)2-CeO2@SNF. It can be seen that the overall structure of SNF remains intact without cracks after the deposition of the nanozyme. The CeO2 and Ni(OH)2 nanozymes are dispersed in the nanochannels of SNF in the form of nanoparticles without obvious agglomeration.

[0075] To further confirm the effective confinement of Ni(OH)2 and CeO2 nanoparticles, high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) technology was further used for characterization. Figure 1 The bright spots shown in E prove the successful deposition of the nanoparticles, Figure 1 and the energy-dispersive X-ray spectroscopy mapping (EDS mapping) image shown in F shows the presence of four elements, Si, O, Ce, and Ni, and the distributions of Ce and Ni elements highly coincide with that of the Si element, further verifying the uniform distribution of the composite nanozyme in the SNF nanochannels.

[0076] Figure 1 Figure G shows the TEM image of the cross-section of Ni(OH)2-CeO2@SNF, revealing the uniform distribution of the composite nanozyme in the ordered nanochannels, indicating that the composite nanozyme is effectively confined within the nanochannels. Figure 1 The high-resolution TEM (HRTEM) image shown in H also clearly shows the lattice structures of the two types of nanoparticles, and the lattice spacing corresponds to the (111) crystal plane of the CeO2 nanoparticles, and the lattice spacing corresponds to the (100) crystal plane of the Ni(OH)2 nanoparticles, which further proves the successful confinement of the Ni(OH)2-CeO2 composite nanozyme in the nanochannels.

[0077] Figure 2 In Figure A are the XPS survey spectra of the electrode obtained by electro-depositing CeO2 on SNF / ITO (CeO2@SNF / ITO) with A being SNF / ITO, and the electrode obtained by successively electro-depositing CeO2 and Ni(OH)2 (Ni(OH)2-CeO2@SNF / ITO). It can be seen that due to the introduction of the SiO2 structure in SNF, characteristic peaks of Si 2p (100.6 eV), O 1s (529.5 eV), and C 1s (284.6 eV) appear in the SNF / ITO electrode. After electro-depositing CeO2, characteristic peaks of the Ce 3d energy level appear in the CeO2@SNF / ITO electrode, and after confining the Ni(OH)2-CeO2 composite nanozyme into the nanochannels through two electro-depositions, characteristic peaks of the Ni 2p energy level further appear.

[0078] Figure 2 In Figure B- Figure 2 In Figure E are the high-resolution Si 2p ( Figure 2 in Figure B), Ce 3d ( Figure 2 in Figure C), Ni 2p ( Figure 2 in Figure D), and O 1s ( Figure 2 in Figure E) spectra of the Ni(OH)2-CeO2@SNF / ITO electrode. The high-resolution Si 2p spectrum proves the existence of the SNF silica structure ( Figure 2 in Figure B). In the high-resolution Ce 3d spectrum, the three characteristic peaks located at 914.0 eV, 901.4 eV, and 898.3 eV (denoted as u1, u2, u3) respectively belong to Ce 4+ 3d 3 / 2 energy level, while the three characteristic peaks located at 895.8 eV, 882.6 eV, and 879.9 eV (denoted as v1, v2, v3) belong to Ce 4+ 3d 5 / 2 energy level ( Figure 2 in Figure C). It is worth noting that peaks located at 904.2 eV and 885.6 eV (denoted as u4, v4) also appear in the high-resolution spectrum, which belong to the characteristic peaks of Ce 3+ . This may be due to the ultra-small nanochannels restricting the growth of the CeO2 nanozyme, making it easier for Ce 3+ to appear on the surface, thus leading to the formation of oxygen vacancies.

[0079] In the Ni 2p spectrum ( Figure 2 in Figure D), the two characteristic peaks located at 878.1 eV and 871.4 eV correspond to the Ni 2+ 2p 1 / 2 energy level in Ni(OH)2, while the two characteristic peaks located at 859.5 eV and 853.2 eV correspond to Ni 2+ 2p3 / 2 Characteristic peaks of energy levels. In addition, the characteristic peak at 529.5 eV in the high-resolution O 1s spectrum belongs to lattice oxygen, mainly from the Si-O bond and Ce-O bond in SiO2 and CeO2. The presence of oxygen vacancies (530.8 eV) was also observed in the spectrum, which is consistent with the information in the Ce 3d spectrum ( Figure 2 in E). At the same time, the OH- characteristic peak at 531.5 eV further proves that Ni exists in the form of Ni(OH)2.

[0080] Figure 3 Figure A in [X] compares the effects of single nanozymes (CeO2 or Ni(OH)2, that is, CeO2 or Ni(OH)2 is electrodeposited alone in step (3) during preparation) and Ni(OH)2-CeO2 composite nanozymes on the electrochemiluminescence intensity of the luminol-dissolved oxygen system. The ECL intensity of the SNF / ITO electrode with unrestricted nanozyme in the luminol-dissolved oxygen system is only 741 a.u., indicating a low luminescence efficiency. After confining CeO2 or Ni(OH)2, the ECL intensity is increased to 7263 a.u. and 5811 a.u. respectively, while the ECL signal of the composite nanozyme (Ni(OH)2-CeO2@SNF / ITO) is significantly enhanced to 20023 a.u., showing excellent synergistic effects. The CV curve shows that the composite nanozyme significantly increases the oxidation peak current of luminol and the reduction peak current of O2, and the reduction potential shifts positively from -0.762 V to -0.194 V, indicating its efficient electrocatalytic performance.

[0081] Figure 3 Figure B in [X] optimized the electrodeposition time of CeO2 or Ni(OH)2. After fixing the electrodeposition time of the CeO2 nanozyme at 10 s, the electrodeposition time of the Ni(OH)2 nanozyme was adjusted; then, conversely, the electrodeposition time of the Ni(OH)2 was fixed at 10 s, and the electrodeposition time of the CeO2 nanozyme was adjusted to investigate the sensitization effect of the composite nanozyme on the ECL intensity. It was found that the ECL signal reached the peak when CeO2 was deposited for 15 s and Ni(OH)2 was deposited for 10 s. An excessive deposition time would lead to nanoparticle aggregation and channel blockage, reducing the catalytic activity.

[0082] Figure 3 Figure C in [X] studied the effect of pH on the ECL intensity of the electrode and found that the Ni(OH)2-CeO2@SNF / ITO electrode showed a relatively high ECL intensity under neutral conditions and was further enhanced under alkaline conditions. Figure 3In D, the ECL signals of the electrodes with and without the SNF-confined composite nanozyme were compared. It was found that the ECL signal significantly decayed with the scanning time without confinement, while the Ni(OH)2-CeO2@SNF / ITO electrode after confinement showed excellent stability and a 27-fold increase in the ECL signal, with an RSD of only 1.1% within 12 cycles. As Figure 3 In E and Figure 3 The continuous ECL measurements in F showed that the electrode signal was stable after the confinement of the composite nanozyme, with an RSD of 1.1%, while the signal of the electrode directly deposited with the composite nanozyme (Ni(OH)2-CeO2@ITO, the preparation method of which was to replace the O-SNF / ITO electrode in step (3) with an ITO electrode and obtain it by electrodeposition through the same steps) significantly decreased with the scanning time.

[0083] Figure 3 As shown in G, under neutral conditions, the ECL intensity of the Ni(OH)2-CeO2@SNF / ITO electrode increased by 35.8 times and 33.7 times compared with SNF / ITO and ITO, respectively, which was attributed to the fact that the nanochannel confinement effect improved the dispersion, active site exposure, and reaction efficiency of the nanozyme.

[0084] As Figure 4 In A and Figure 4 As shown in B, the electrochemical behavior and ECL intensity of the Ni(OH)2-CeO2@SNF / ITO electrode under different atmospheres. As the dissolved oxygen content increased, the O2 reduction peak current and the ECL signal increased synchronously; while under N2 saturation conditions, the ECL signal almost disappeared, indicating that O2 was a necessary condition for the generation of ROS.

[0085] Figure 4 The free radical capture experiment of the Ni(OH)2-CeO2@SNF / ITO electrode in the PBS buffer solution containing luminol shown in C verified that after adding the O2 ·- scavenger BQ, the ECL signal was almost completely quenched, while the OH· scavenger TBA had no significant effect on the signal, indicating that O2 ·- was the main ROS. Figure 4 The EPR experiment shown in D further confirmed the existence of O2 ·- and the amount of O2 generated by the composite nanozyme electrode was significantly higher than that of the single nanozyme electrode, reflecting the synergistic catalytic effect. ·-

[0086] As Figure 4 In E- Figure 4As shown in Fig. F, the electrochemically scanning direction was changed (starting from -0.2 V and scanning towards the anode and cathode respectively) to explore the sensitization mechanism of the composite nanozyme on the ECL of the luminol-dissolved oxygen system. When the SNF / ITO electrode was scanned anodically, an ECL signal (704 a.u.) appeared only in the fifth cycle, while the Ni(OH)2-CeO2@SNF / ITO electrode generated a signal of 997 a.u. in the first cycle, indicating that the composite nanozyme significantly increased the production of L ·- . During cathodic scanning, the difference in the ECL signals of the two electrodes in the first and fifth cycles was small, indicating that O2 ·- had a long lifetime and could react with L ·- to produce anodic luminescence. The electrode modified with the composite nanozyme had a stronger ECL signal, which was attributed to its catalysis of the reduction of O2 to generate more ROS, significantly enhancing the ECL intensity.

[0087] As Figure 4 shown in Fig. G, under the condition of the same reaction time and using H2O2 as the substrate, the electrodes confined with the two single nanozymes led to an increase in the absorbance value of the solution, indicating that both Ni(OH)2 and CeO2 had good peroxidase activity, and the electrode co-confined with the two nanozymes showed better activity. As Figure 4 shown in Fig. H, the enzymatic reactions using O2 as the substrate showed that the electrodes modified with single nanozymes or composite nanozymes all exhibited good oxidase (OXD) activity. Therefore, the composite nanozyme synthesized by the electrochemical deposition method not only had POD activity but also OXD activity.

[0088] The effective electroactive area of the electrode before and after the confinement of the composite nanozyme was measured using the standard electrochemical probe ferrocenemethanol according to the Randles-Sevcik equation.

[0089] I p =(2.69×10 5 )AD 1 / 2 n 3 / 2 v 1 / 2 c

[0090] where D is the diffusion coefficient of FcMeOH (cm 2 ·s -1 ), n is the number of electrons transferred during the redox reaction (n = 1), and c is the concentration of FcMeOH (c = 0.5 mM).

[0091] As Figure 5 shown in Fig. A- Figure 5As shown in B of Example 1, in a 0.1 M KCl solution containing 0.5 mM FcMeOH, the CV curves of SNF / ITO and Ni(OH)2-CeO2@SNF / ITO at different scan rates (20 mV / s, 40 mV / s, 60 mV / s, 80 mV / s, 100 mV / s, 120 mV / s, 140 mV / s) are shown. The inset is the linear regression curve between the CV peak current and the square root of the scan rate. The oxidation peak current (I p ) and the square root of the CV scan rate (v 1 / 2 ) show a good linear relationship. The linear equations corresponding to the two modified electrodes are I p1 = 87.2v 1 / 2 + 0.09 (k1 = 87.2, R 2 = 0.999) and I p2 = 69.9v 1 / 2 + 3.17 (k1 = 87.2, R 2 = 0.997). The ratio of the electroactive areas of the two modified electrodes can be expressed by the following equation:

[0092]

[0093] It can be seen that the effective electroactive area of the electrode after confinement of Ni(OH)2-CeO2 decreases, which is attributed to the increase in the interfacial resistance caused by the introduction of oxides and hydroxides. Therefore, the significant increase in the ECL signal after modification with the composite nanozyme is not due to the increase in the effective electroactive area.

[0094] As Figure 5 shown in C- Figure 5 D, in a 0.01 M PBS solution (pH = 7.4) containing dissolved oxygen, compared with the SNF / ITO electrode, the Faraday current of the Ni(OH)2-CeO2@SNF / ITO electrode at 0.8 V increases significantly, reaching 2.85 μA, which is attributed to the oxidation of the Ni(OH)2-CeO2 composite nanozyme. In addition, the reduction peak potential of O2 shifts positively, and the reduction peak current increases, indicating that the Ni(OH)2-CeO2 composite nanozyme exhibits excellent activity in the electrocatalytic reduction process of O2. After purging with N2 to remove most of the O2, these phenomena are significantly weakened.

[0095] As Figure 5 shown in E- Figure 5As shown in Fig. F, when luminol was added, the Faraday current of the SNF / ITO electrode at 0.8 V was 1.45 μA. Interestingly, in the presence of the Ni(OH)2-CeO2 composite nanozyme, at the same potential (0.8 V), the Faraday current increased to 8.82 μA. After subtracting the oxidation current of the Ni(OH)2-CeO2 composite nanozyme itself (2.85 μA), the Faraday current of luminol at 0.8 V on the Ni(OH)2-CeO2@SNF / ITO electrode was 5.97 μA. This indicates that the Ni(OH)2-CeO2 composite nanozyme significantly enhanced the Faraday current of luminol at 0.8 V, increasing by approximately 4.1 times (5.97 μA / 1.45 μA). The above results suggest that the Ni(OH)2-CeO2 composite nanozyme sensitizes the luminol-O2 ECL through two pathways: (1) catalytically oxidizing luminol to generate L ·- , (2) catalytically reducing O2 to generate O2 ·- .

[0096] The ECL reaction mechanism of the luminol + dissolved oxygen + composite nanozyme system is as follows:

[0097] (1) Cathodic scanning process:

[0098] Ce(Ⅳ)+e - →Ce(Ⅲ)

[0099] 3O2+2H2O+3Ce(Ⅲ)+3Ni(Ⅱ)→3Ce(Ⅳ)+3Ni(Ⅲ)+2O2 ·- +4OH -

[0100] O2+2H2O+Ce(Ⅲ)+Ni(Ⅱ)→Ce(Ⅳ)+Ni(Ⅲ)+H2O2+2OH -

[0101] Ni(Ⅲ)+e - →Ni(Ⅱ)

[0102] (2) Anodic scanning process:

[0103] 2H2O2+4OH - +Ce(Ⅳ)+Ni(Ⅲ)→2O2 ·- +Ce(Ⅲ)+Ni(Ⅱ)+4H2O

[0104] Ce(Ⅲ)–e - →Ce(Ⅳ)

[0105] Ni(Ⅱ)–e - →Ni(Ⅲ)

[0106] LH2–H + →LH-

[0107] LH -- 2e - →L ·- +H +

[0108] L ·- +O2 ·- →LO2 2-

[0109] LO2 2- →AP 2- *+N2

[0110] AP 2- *→AP 2- +hν

[0111] Ni(OH)2-CeO2 catalyzes the reduction of O2 to generate O2 through a dual-cycle catalytic mechanism ·- , significantly enhancing ECL. During the cathodic scan, Ce(IV) is reduced to Ce(III), which synergistically catalyzes the conversion of O2 to O2 ·- and H2O2 with Ni(II). Meanwhile, Ce(III) is re-oxidized to Ce(IV), and Ni(III) is reduced to Ni(II) at the electrode interface, maintaining the catalytic cycle. During the anodic scan, Ce(IV) and Ni(III) catalyze the oxidation of H2O2 to generate more O2 ·- , and are reduced to Ce(III) and Ni(II), which are re-oxidized to Ce(IV) and Ni(III), continuously catalyzing the reaction. Meanwhile, luminol is anodically oxidized to form LH−, which further loses protons and electrons to form L ·- , which reacts with O2 ·- to generate LO2 2- , which is finally converted to the excited state AP2−*, emitting an ECL signal.

[0112] As Figure 6 shown in A- Figure 6 and C in TNF-α ((ⅰ: SNF / ITO, ⅱ: O-SNF / ITO, ⅲ: Ab TNF-α / O-SNF / ITO, ⅳ: BSA / Ab TNF-α / O-SNF / ITO, ⅴ: TNF-α / BSA / Ab 3- / 4- / O-SNF / ITO)); After the long-range ordered nanochannel array of SNF modifies the electrode, [Fe(CN)6] on the SNF / ITO electrode 3- / 4-A clear and symmetric pair of redox peaks was exhibited, demonstrating good mass transfer performance of the electrode. After covalent bonding of the epoxy group to the SNF surface, the CV redox peak current decreased slightly, which might be attributed to the influence of surface epoxy group functionalization on the surface charge and interfacial resistance of the electrode. During the electrode modification process, the coupling of the biorecognition molecule (antibody, Ab) and the addition of the blocking protein BSA further significantly reduced the electrochemical signal intensity, which was attributed to the fact that proteins, as large-sized insulating molecules, increased the interfacial resistance on the one hand and caused steric hindrance on the other hand, reducing the diffusion of small molecule probes to the substrate electrode.

[0113] Further DPV analysis showed that the decrease in the redox peak current was consistent with the increase in the charge transfer resistance (Rct), verifying the significant change in the interfacial properties during the electrode modification process. These results indicated that the antibody had been successfully modified onto the electrode surface, demonstrating the successful construction of the immunosensing interface. In addition, when the target was present, the specific binding of the antigen (Ag) to the antibody led to the formation of an immune complex on the outer surface of the SNF, further increasing the interfacial resistance and steric hindrance, manifested as a significant decrease in the CV and DPV signals and a further increase in Rct in EIS. Therefore, due to the specific recognition of the target and the formation of the immune complex, the increase in the interfacial steric hindrance inhibited the mass transfer process of the signal probe, enabling the detection of target molecules based on the "signal-off" mode.

[0114] As Figure 6 shown in D, where i: Ni(OH)2-CeO2@SNF / ITO, ii: Ni(OH)2-CeO2@O-SNF / ITO, iii: Ab TNF-α / Ni(OH)2-CeO2@O-SNF / ITO, iv: BSA / Ab TNF-α / Ni(OH)2-CeO2@O-SNF / ITO, v: TNF-α / BSA / Ab TNF-α / Ni(OH)2-CeO2@O-SNF / ITO;

[0115] In the luminol-dissolved oxygen system, the Ni(OH)2-CeO2@SNF / ITO electrode showed excellent ECL signals. However, with the modification of the epoxy functional group and Ab, the ECL signals gradually weakened. When TNF-α was specifically recognized and captured by its antibody, the formation of the immune complex further reduced the ECL signal. These results fully demonstrated the successful construction of the ECL immunosensor, the specific capture of target molecules, and the feasibility of detection.

[0116] Meanwhile, two key parameters, the incubation time of antibody Ab and the incubation time of antigen Ag, were systematically optimized to achieve the best sensing performance. As Figure 6 shown in E andFigure 6 As shown in Figure F, when the antibody incubation time exceeds 90 min, the ECL signal gradually stabilizes, indicating that most of the Abs have been effectively immobilized on the electrode surface. Similarly, when the incubation time of TNF-α exceeds 60 min, the signal reaches a steady state, suggesting that the antigen-antibody binding has reached saturation. Considering factors such as time cost, the optimal Ab incubation time was determined to be 90 min, while the optimal incubation time for TNF-α was 60 min.

[0117] As Figure 7 shown in Figure A- Figure 7 As shown in Figure B, with the increase in the concentration of TNF-α (a: 0 pg / mL; b: 0.01 pg / mL; c: 0.1 pg / mL; d: 1 pg / mL; e: 10 pg / mL; f: 100 pg / mL; g: 1 ng / mL; h: 10 ng / mL.), where the reproducibility of the sensor was evaluated by detecting TNF-α at the same concentration (1 ng / mL) multiple times ( Figure 7 Figure C). The results showed that the relative standard deviation (RSD) of the ECL signals of five parallel-prepared immunosensors for detecting 1 ng / mL TNF-α was no more than 2.0%, further indicating that the electrode had good reproducibility and also confirming the repeatability of the sensor preparation process. To comprehensively evaluate the performance of the prepared immunosensor, its detection selectivity, anti-interference ability, and storage stability were systematically studied.

[0118] Other tumor markers (including IL-6, CEA, CA199, CA242, PCT), small molecules commonly found in biological fluids (such as glucose Glu, uric acid UA), and common inorganic ions (such as Na + , Cl - ) were used as potential interferents, with the concentration of TNF-α being 1 ng / mL, the concentration of CA199 and CA242 being 10 U / mL, the concentration of IL-6, CEA, and PCT being 10 ng / mL, and the concentration of UA, Glu, Na + and Cl - being 100 μM; the selectivity and anti-interference ability of the sensor were evaluated by the degree of decrease in the ECL signal of the electrode in the presence of these substances ( Figure 7 Figure D). The results showed that the ECL signal of the immunosensor decreased significantly only in the presence of the target analyte TNF-α, while other substances did not significantly affect the electrode signal. This indicates that the sensor has high selectivity for TNF-α and exhibits good anti-interference ability against common interferents. This performance can be attributed to the high specific recognition ability of the antibody in the sensor. In addition, after storing the immunosensor at 4 °C for 6 days, its detection ability for 1 ng / mL TNF-α did not show significant changes, and the RSD of the relative ECL signal during storage was 1.2% (Figure 7 In (E), the error bars represent the standard deviation of three measurements. This result indicates that the sensor has high storage stability.

Claims

1. A confined composite nanozyme enhanced electrochemiluminescence immunosensor for detecting tumor necrosis factor, characterized in that, The immunosensor is a three-electrode system, and the liquid to be detected contains luminol. The working electrode is obtained by covalently immobilizing a tumor necrosis factor-specific antibody on a SNF epoxy-modified electrode after confining Ni(OH)2-CeO2 composite nanozyme and then blocking non-specific sites.

2. The electrochemiluminescence-enhanced immunosensor for detecting tumor necrosis factor with a confined composite nanozyme according to claim 1, wherein The preparation of the working electrode includes the steps: Step 1, removing micelles from the micelle-containing SNF modified electrode after epoxy silane modification; Step 2, electro-depositing the electrode obtained in Step 1 in a Ce precursor solution and then in a Ni precursor solution in sequence to obtain an electrode with pore-confined Ni(OH)2-CeO2 composite nanozyme; Step 3, incubating the electrode obtained in Step 2 in a buffer solution containing tumor necrosis factor antibody, rinsing, and then incubating in a BSA solution to obtain the working electrode.

3. The electrochemiluminescence enhanced immunosensor for detecting tumor necrosis factor with a confined composite nanozyme according to claim 2, wherein The specific steps of Step 1 include: soaking the micelle-containing SNF modified electrode in a solution containing epoxy group silane, and then immersing it in an ethanol solution containing inorganic acid to remove micelles.

4. The electrochemiluminescence enhanced immunosensor for detecting tumor necrosis factor with a confined composite nanozyme according to claim 3, characterized in that The epoxy group silane includes any one of γ-methacryloyloxypropyltrimethoxysilane, 2-(3,4-epoxycyclohexyl)ethyltriethoxysilane, and epoxy group polyethylene glycol silane; the solvents used include any one or more of ethanol, methanol, acetone, cyclohexane, and acetonitrile; the molar concentration of the solution containing epoxy group silane is 2-5 mmol / mL; The electrode is soaked in the solution containing epoxy group silane at room temperature for 0.3-3 h; The molar concentration of inorganic acid in the ethanol solution containing inorganic acid is 0.01-1 mol / mL, and the electrode is stirred in the ethanol solution containing inorganic acid for 1-30 min.

5. The electrochemiluminescence enhanced immunosensor for detecting tumor necrosis factor with a confined composite nanozyme according to claim 2, wherein The Ce precursor solution includes a solution of any one or more of cerium nitrate, cerium sulfate, and cerium chloride, and the solvents used include any one or more of deionized water, sodium chloride aqueous solution, potassium chloride aqueous solution, phosphate buffer solution, or Tris-HCl buffer solution; The molar concentration of cerium salt in the solution of the Ce precursor is 0.01-0.5 mol / L; The Ni precursor solution includes a solution of any one or more of nickel chloride, nickel sulfate, and nickel nitrate, and the solvents used include any one or more of deionized water, sodium chloride aqueous solution, potassium chloride aqueous solution, phosphate buffer solution, or Tris-HCl buffer solution; The molar concentration of nickel salt in the solution of the Ni precursor is 0.01-0.2 mol / L.

6. The electrochemiluminescence-enhanced immunosensor for detecting tumor necrosis factor with a confined composite nanozyme according to claim 2, wherein In Step 2, the constant potential range for electro-depositing the electrode in the Ce precursor solution is -1.0 to -0.3 V, the electro-depositing time is 2 to 360 s, and the temperature is room temperature; The constant potential range for electrodeposition of the electrode in the Ni precursor solution is -0.02 to -0.3 mA / cm 2 , the electrodeposition time is 2 to 120 s, and the temperature is room temperature.

7. The electrochemiluminescence enhanced immunosensor for detecting tumor necrosis factor with a confined composite nanozyme according to claim 2, wherein In Step 3, the mass concentration of tumor necrosis factor antibody in the buffer solution containing tumor necrosis factor antibody is 5-30 μg / mL; the solvents used include any one or more of STE buffer solution, TE buffer solution, or phosphate buffer solution; the solute concentration in the buffer solution is 0.005-0.5 mol / L, and the pH is 6.5-7.5; And / or, the incubation condition in the buffer solution containing tumor necrosis factor antibody in Step 3 is incubation at 0-5 °C for 0.5-5 h; the incubation condition in the BSA solution is incubation at room temperature for 5-30 min.

8. The method for constructing an immunosensor according to any one of claims 1-7, characterized in that, It includes the steps: Step 1: Remove the micelles after modifying the micelle-containing SNF modified electrode with epoxy silane; Step 2: Electrochemically deposit the electrode obtained in Step 1 in a Ce precursor solution and a Ni precursor solution in sequence to obtain an electrode with pore-confined Ni(OH)2-CeO2 composite nanozyme; Step 3: Incubate the electrode obtained in Step 2 in a buffer solution containing tumor necrosis factor antibody, rinse it, and then incubate it in a BSA solution to obtain a working electrode; Step 4: Use the working electrode to form a three-electrode system with a reference electrode and a counter electrode, and perform electrochemiluminescence signal detection on the test solution containing luminol.

9. The method for detecting tumor necrosis factor using the immunosensor according to any one of claims 1-7, characterized in that, It includes the steps of: incubating the working electrode in the test solution at 0-5°C for 0.5-5 h, rinsing it, and then performing electrochemiluminescence signal detection; the pH of the test solution is 6.5-10, and the molar concentration of luminol is 20-180 μmol / L.

10. The method for detecting tumor necrosis factor by the immunosensor according to claim 9, characterized in that, The test solution further includes any one or more interfering proteins among bovine serum albumin, alpha-fetoprotein, and carcinoembryonic antigen, and the concentration of each interfering protein is below 10 ng / mL; the solution environment of the test solution is Tris buffer or phosphate buffer.

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