Immunosensor for detecting cytokine interferon gamma through electrochemiluminescence as well as preparation method and application of immunosensor

Abstract

The invention discloses an immunosensor for detecting cytokine interferon gamma through electrochemiluminescence as well as a preparation method and application of the immunosensor. The immunosensor comprises a three-electrode system consisting of an immunoelectrode, a reference electrode and a counter electrode, the immunoelectrode is a VMSF modified electrode confined to cerium dioxide nano enzyme and platinum nano particles, and is obtained by covalently immobilizing an IFN-gamma antibody and closing a non-specific site; when the immunosensor is used for detection, a solution containing luminol is used as an electrolyte. The CeO2 and the PtNPs which are confined in the nano channel show a remarkable synergistic catalysis effect. Compared with direct modification on a plate electrode, CeO2-PtNPs has higher catalytic activity and stability. The sensor provided by the invention is used for INF-gamma detection, has the advantages of high sensitivity, low detection limit and wide detection range, does not need complex pretreatment and separation processes during detection, reduces the detection cost, is simple to operate, and is suitable for large-scale application.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to an immunosensor for detecting cytokine interferon gamma through electrochemiluminescence, and a preparation method and application thereof. Background Art

[0002] Cytokines are a class of small proteins or peptides secreted by cells that participate in key biological processes such as immune regulation, inflammatory responses, cell differentiation, and signal transduction. Abnormal cytokine expression is closely associated with the development and progression of various diseases. Accurate cytokine detection not only provides insights into disease pathogenesis but also facilitates early diagnosis and disease progression monitoring. For example, interferon gamma (IFN-γ) testing holds significant clinical significance in obstetrics, primarily by reflecting inflammatory responses, immune imbalance, and vascular dysfunction at the maternal-fetal interface, providing a basis for the prediction, diagnosis, and monitoring of various pregnancy complications. Specifically, significantly elevated levels of IFN-γ and its induced protein, IFI16, are associated with the development, progression, and severity of preeclampsia and can predict its risk. In premature rupture of membranes (PROM), activation of the IFN-γ pathway and its association with inflammatory cytokines (such as IL-6 and IL-1β) suggest a role in the pathogenesis of infectious PROM, facilitating early diagnosis. Elevated umbilical cord blood serum IFN-γ levels are an independent risk factor for preterm birth. Their synergistic effect with proinflammatory cytokines (such as IL-12p70 and IL-17A) reflects an intrauterine inflammatory state. In gestational diabetes mellitus (GDM), elevated serum IFN-γ levels and an increased IFN-γ / IL-4 ratio are closely associated with adverse perinatal outcomes (such as asphyxia and malformations), and combined testing can enhance predictive value. Furthermore, in assisted reproduction, abnormally elevated IFN-γ levels combined with decreased progesterone levels can predict the risk of biochemical pregnancy or miscarriage. Luteal support therapy, which modulates the Th1 / Th2 balance (inhibiting IFN-γ and promoting IL-4), can help improve pregnancy outcomes. Therefore, IFN-γ testing is an important tool for assessing the risk and management of obstetric complications.

[0003] Currently, commonly used IFN-γ detection methods include enzyme-linked immunosorbent assay (ELISA), flow cytometry, and Luminex technology multiplex analysis. However, ELISA is a commonly used method for single cytokine detection, but the operation steps are cumbersome and the sensitivity needs to be improved. Flow cytometry can perform multiplex detection of multiple cytokines at the single-cell level, with the advantages of high throughput and high resolution. However, its equipment is expensive and the operation is complicated. Luminex technology has high requirements for sample processing and high equipment costs. Therefore, it is of great significance to develop novel, convenient, and highly sensitive cytokine detection methods.

[0004] ECL offers advantages such as low background, high signal-to-noise ratio, and high analytical sensitivity, and holds great potential in numerous fields, including immunoassays, environmental testing, food safety, drug analysis, and public safety. The luminescent reagent luminol has attracted considerable attention due to its unique properties, including low toxicity and high storage stability. The traditional co-reactant, H2O2, can significantly enhance the electrochemiluminescence (ECL) signal of luminol. However, its poor room temperature stability and high biotoxicity limit the application of luminol-H2O2-based ECL systems in biosensing. The endogenous co-reactant, dissolved oxygen (DO), is stable and non-toxic at room temperature and pressure, making it an ideal co-reactant for luminol. Luminol-DO-based ECL systems typically exhibit high luminescence intensity only under alkaline conditions; strong ECL signals are difficult to observe under near-neutral conditions. This is attributed to the low efficiency of DO conversion to reactive oxygen species (ROS) under near-neutral conditions. Therefore, improving the luminescence efficiency of luminol-DO ECL systems under near-neutral conditions is crucial for constructing highly sensitive ECL sensors.

[0005] Recent research has shown that the detection performance of ECL systems can be improved by introducing a coreaction accelerator, constructing an ECL ternary system consisting of a luminophore / coreaction reagent / coreaction accelerator. Coreaction accelerators interact with coreaction reagents, increasing the number of coreaction reagent free radicals produced per unit time by the electrochemical reaction. This interaction between free radicals significantly enhances the luminescence intensity of the luminescent substance.

[0006] For example, CN110455786 A discloses a preparation method for a CeO2@SnS2-promoted luminol electrochemiluminescence sensor. Based on the electrochemiluminescence ECL technology, ferritin covalently cross-linked luminol (Ft-luminol) is used as a signal source for the first time, and CeO2@SnS2 is used as a promoter. The excellent synergistic catalytic effect of CeO2@SnS2 on luminol is utilized to effectively amplify the detection signal. A simple, low-cost, low-reaction energy consumption, and green and environmentally friendly biosensor preparation method is proposed, and it is applied to the actual sample detection of procalcitonin. The detection limit is as low as 1.6 fg / mL, the linear range is as wide as 5 fg / mL-100 ng / mL, the sensitivity is high, the reproducibility is good, and it has great potential application value.

[0007] CN109490284 A discloses a dual-catalytic luminol electrochemiluminescence biosensor based on gold nanoparticles and titanium dicarbide MXenes. Combining a multi-site recognition strategy with the reducibility of a novel two-dimensional material, Ti3C2MXenes, AuNPs-MXenes are used as ECL signal probes. This allows for the design of a highly sensitive electrochemiluminescence sensing platform for detecting HeLa exosomes. In this strategy, SA polymer molecules provide a multi-site recognition interface to capture more aptamers, thereby increasing exosome capture efficiency. This helps improve the sensitivity of the biosensor, and the AuNPs-MXenes dually catalyze the luminol ECL signal enhancement. Based on these two dual amplification strategies, a highly sensitive and selective biosensor for detecting exosomes was obtained. Furthermore, the ECL biosensor has also been used for exosome detection in serum.

[0008] Recent studies have shown that some noble metal and metal oxide nanomaterials exhibit significant catalytic activity for the electrochemical reduction of dissolved oxygen. However, these materials are prone to detachment when directly loaded onto electrodes, resulting in low stability. Confining these materials within porous materials can improve their stability and activity. Summary of the Invention

[0009] To address the problems of low sensitivity and difficulty in detecting the cytokine IFN-γ in serum samples, the present invention provides a nanochannel-confined electrochemiluminescence sensor for detecting cytokines. Confined cerium dioxide (CeO2) and platinum nanoparticles (CeO2-PtNPs) are used as co-reactant promoters to construct a label-free immunosensor, which can achieve high-sensitivity ECL detection of cytokines.

[0010] To achieve the above object, the technical solution adopted by the present invention is: An immunosensor for electrochemiluminescence detection of cytokine interferon-γ, the immunosensor comprising a three-electrode system of an immunoelectrode, a reference electrode, and a counter electrode; The immunoelectrode is a vertically ordered mesoporous silica film (VMSF) modified electrode that confines ceria nanozymes and platinum nanoparticles, obtained by covalently fixing IFN-γ antibodies and blocking nonspecific sites; The immunosensor uses a solution containing luminol as an electrolyte during detection.

[0011] The present invention uses luminol and antibodies against the cytokine IFN-γ as electrochemiluminescent signal molecules and specific recognition elements, respectively; combines a CeO2-PtNPs composite material confined within a vertically ordered mesoporous silica nanochannel film to enhance the luminol-dissolved oxygen ECL signal, and constructs an immunosensor; as an ECL system, inexpensive luminol is used as the ECL luminogen, and dissolved oxygen is used as an endogenous co-reactant, thereby improving the convenience of detection and reducing detection costs.

[0012] Using CeO2-PtNPs composite materials confined in mesoporous nanochannels as co-reactant promoters, the ECL signal is significantly sensitized and the detection sensitivity is improved. It is used for high-sensitivity detection of IFN-γ, with the advantages of low detection limit and wide detection range. In addition, the detection does not require complex pretreatment and separation processes, which reduces the detection cost, is simple to operate, and is suitable for large-scale applications.

[0013] The catalytic activity of CeO2 nanozymes stems from their redox properties mediated by the reversible conversion of Ce(III) / Ce(IV) oxidation states. In an ECL system with dissolved oxygen, CeO2 nanozymes, acting as co-reaction promoters, catalyze the reduction of O2 to produce high concentrations of reactive oxygen species (ROS), significantly enhancing the ECL signal intensity. PtNPs can catalyze the electrochemical reduction of O2 to produce reactive oxygen species. The composite of CeO2 and PtNPs exhibits a synergistic catalytic effect, further enhancing the ECL response of luminol-O2 systems.

[0014] The immunoelectrode preparation specifically includes the following steps: Step 1: immerse the VMSF modified electrode in a cerium salt solution and electrodeposit to obtain a confined CeO2 electrode; Step 2, immersing the confined CeO2 electrode in a H2PtCl6 solution for electrodeposition to obtain a confined CeO2-PtNPs electrode; Step 3, immersing the confined CeO2-PtNPs electrode in an organosiloxane solution containing epoxy groups and allowing it to react to obtain an epoxy VMSF functionalized electrode; Step 4: immerse the electrode obtained in step 3 in a buffer solution containing IFN-γ antibody and incubate it, and then immerse it in a buffer solution containing BSA and incubate it to obtain the immunoelectrode.

[0015] In this study, CeO2-PtNPs are confined within the porous material of VMSF. Using porous materials to confine nanomaterials significantly improves their stability and activity. Furthermore, the porous material provides dispersion space, effectively increasing the active sites of the nanomaterials, thereby enhancing their catalytic activity and stability.

[0016] The cerium salt includes any one or more of Ce(NO3)3·6H2O, Ce(NH4)2(NO3), and CeCl3; the organosiloxane includes any one or more of γ-glycidyloxypropyltrimethylsilane (GPTMS), 3-(2,3-epoxypropyloxy)propyltrimethoxysilane, and 2-(3,4-epoxycyclohexyl)ethyltriethoxysilane; The solvent used in the cerium salt solution is water; the solvent used in the H2PtCl6 solution is water or sulfuric acid; the solvent used in the organosiloxane solution includes any one or more of acetone, ethanol, methanol, and cyclohexane. The buffer solution includes a sodium chloride aqueous solution, a sodium sulfate aqueous solution, a potassium chloride aqueous solution or a phosphate buffer solution; the solute concentration in the buffer solution is 0.005 to 0.5 mol / L and the pH is 6 to 8.

[0017] The VMSF modified electrode is any one of a VMSF modified indium tin oxide electrode, a glassy carbon electrode, a fluorine-doped tin oxide electrode, a gold electrode, a screen-printed electrode, a graphite electrode, and a carbon fiber electrode; The preparation method of the VMSF modified electrode includes any one of electrochemically assisted self-assembly, two-phase layered growth, evaporation-induced self-assembly, π-π interaction induction, epitaxial growth, high magnetic field, electric field, and organic solvent-induced self-assembly.

[0018] The molar concentration of the cerium salt in the cerium salt solution in step 1 is 0.1 to 0.2 M, the electrodeposition time is 10 to 20 s, and the electrodeposition voltage is -0.7 to -0.8 V; The H2PtCl6 concentration in the H2PtCl6 solution in step 2 is 0.1-1.0 μM, the electrodeposition time is 100 ~120 s, and the electrodeposition voltage is -0.4 V ~ 0.2 V.

[0019] In step 3, the molar concentration of the organosiloxane in the organosiloxane solution is 2.0 mM to 3.0 mM, and the reaction is allowed to stand for 30 to 60 minutes at a temperature of 20 to 40°C. In step 4, the electrode is incubated in the buffer solution containing IFN-γ antibody at 0 to 5°C for 1 to 2 hours; in the buffer solution containing BSA, the electrode is incubated at room temperature for 5 to 30 minutes; The concentration of the IFN-γ antibody in the buffer solution containing the IFN-γ antibody in step 4 is 1 to 20 μg / mL.

[0020] Preferably, each step is rinsed with water or buffer to remove excess undeposited or unincubated solution; The molar concentration of luminol in the detection solution is 5 to 300 μmol / L.

[0021] The present invention also provides a method for preparing an immunosensor for detecting the cytokine interferon-γ by electrochemiluminescence, comprising the steps of: Step 1: immerse the VMSF modified electrode in a cerium salt solution and electrodeposit to obtain a confined CeO2 electrode; Step 2, immersing the confined CeO2 electrode in a H2PtCl6 solution for electrodeposition to obtain a confined CeO2-PtNPs electrode; Step 3, immersing the confined CeO2-PtNPs electrode in an organosiloxane solution containing epoxy groups and allowing it to react to obtain an epoxy VMSF functionalized electrode; Step 4: immerse the electrode obtained in step 3 in a buffer solution containing IFN-γ antibody and incubate it, and then immerse it in a buffer solution containing BSA and incubate it to obtain the immunoelectrode.

[0022] Step 5: forming a three-electrode system with the immunoelectrode, the counter electrode and the reference electrode to obtain the immunosensor, wherein the immunosensor uses a solution containing luminol as an electrolyte during detection.

[0023] The present invention also provides applications of the electrochemiluminescence immunosensor for detecting the cytokine interferon gamma for non-diagnostic or non-therapeutic purposes, such as studying the mechanisms of anti-tumor immune responses and detecting residual tumor markers after drug action.

[0024] The present invention discloses a method for detecting cytokine interferon gamma by electrochemiluminescence. The method comprises the following steps: incubating the immunoelectrode with a solution to be tested and then rinsing to obtain the electrode to be tested; using the electrode to be tested as a working electrode, a reference electrode, and a counter electrode to form a three-electrode system; and using an electrolyte solution containing luminol as a detection solution to perform electrochemiluminescence signal detection.

[0025] The detection principle is as follows: When IFN-γ is present in a serum sample, IFN-γ antibodies bind to IFN-γ. The large immune complex formed at the electrode interface significantly hinders the diffusion and mass transfer of luminol, thereby changing the electrode's ECL signal. Therefore, based on the degree of quenching of the ECL signal, ECL detection of IFN-γ can be achieved.

[0026] The concentration of cytokine interferon γ in the test solution is below 100 ng / mL; The incubation is carried out at 0-5°C for 5-120 minutes.

[0027] The solution to be tested includes one or more interfering compounds selected from glucose, inorganic salts, cytokines, and tumor markers; and the total molar concentration of the interfering compounds is below 0.5M.

[0028] Preferably, the sample solution to be tested includes a standard solution containing the cytokine interferon γ or a serum sample.

[0029] The detection limit of the electrochemiluminescence detection method reached 4.2 fg / mL.

[0030] Compared with the prior art, the present invention has the following beneficial effects: (1) The electrochemiluminescence immunosensor of the present invention uses luminol as the ECL luminogen, dissolved oxygen as the endogenous co-reactant, and the CeO2-PtNPs composite material confined in the mesoporous nanochannel as the co-reactant promoter, which significantly enhances the ECL signal and improves the detection sensitivity. It is used for the highly sensitive detection of IFN-γ in serum samples and has the advantages of a wide linear range, high selectivity, high detection sensitivity, and a low detection limit.

[0031] (2) In the present invention, CeO2-PtNPs are placed in nanochannels. Both materials are ultra-small in size, and the two nanomaterials produce a synergistic catalytic effect, which significantly enhances the signal sensitization of the luminol-dissolved oxygen system compared to a single material.

[0032] (3) In the present invention, the CeO2-PtNPs composite material confined in the nanochannel can effectively avoid its ineffective diffusion by in-situ catalytic generation of ROS on the inner surface of the nanochannel, thereby increasing its local concentration and thus improving the reaction efficiency.

[0033] (4) The CeO2-PtNPs composite material confined in the nanochannel of the present invention shows higher catalytic activity and higher stability than the nanomaterial directly deposited on the electrode.

[0034] (5) The silica nanoporous membrane of the present invention has ultra-small nanochannels (2~3 nm) and the surface of the pores is rich in silanol groups. It can hinder the influence of large-sized negatively charged pollutants (such as cells, proteins and other biological macromolecules) in complex samples on the base electrode through electrostatic repulsion, hydrophilic interaction and size screening effect, and has good anti-fouling and anti-interference capabilities. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the microscopic morphology of the VMSF-modified ITO electrode (VMSF / ITO) in Example 1. Figure 1 (A) is a scanning electron microscope (SEM) image of the cross section of VMSF-modified ITO electrode (VMSF / ITO). Figure 1Middle (B) is a top-view transmission electron microscope (TEM) image of VMSF / ITO, and the inset is a high-resolution TEM image. Figure 1 Middle (C) is the TEM image of the cross section of the VMSF / ITO electrode.

[0036] Figure 2 is the XPS characterization data of different electrodes in Example 1, Figure 2 (A) is the XPS full spectrum scan of VMSF / ITO, CeO2@VMSF / ITO and CeO2-PtNPs@VMSF / ITO electrode surfaces. Figure 2 Middle (B) is the XPS high-resolution spectrum of O 1s energy level on the surface of CeO2-PtNPs@VMSF / ITO electrode. Figure 2 Middle (C) is the XPS high-resolution energy spectrum of Ce 3d energy level on the surface of CeO2-PtNPs@VMSF / ITO electrode. Figure 2 Middle (D) is the XPS high-resolution energy spectrum of Pt 4f energy level on the surface of CeO2-PtNPs@VMSF / ITO electrode.

[0037] Figure 3 ECL intensity-time curves of the electrodes obtained by electrodeposition of CeO2, PtNPs, and CeO2-PtNPs on ITO electrode and VMSF / ITO electrode, respectively, in 0.01 M PBS (pH = 7.4) containing 100 μM luminol. Figure 3 (A) in the middle is the ITO electrode. Figure 3 Middle (B) is VMSF / ITO.

[0038] Figure 4 The electrochemical behavior and ECL intensity changes of CeO2-PtNPs@VMSF / ITO electrode in Example 1 under different atmospheres are shown. Figure 4 (A) is the CV curve of the electrode under different atmospheres. Figure 4 Middle (B) is the ECL signal of the electrode under different atmospheres. Figure 4 (C) is the ECL potential diagram of the electrode in 0.01 M PBS (pH = 7.4) containing 100 μM luminol when the starting scan potential is 0 V and the initial scan direction is positive (left) and negative (right). Figure 4 (D) shows the ECL response of the electrode in the luminol-O2 system after adding different free radical scavengers. Figure 4 Middle (E) shows the UV-visible absorption spectra of different electrodes after being inserted into 0.1 M NaAc / HAc buffer solution (pH 4.0) containing 0.25 mM TMB and 1 mM H2O2 for 20 min. Figure 4Middle (F) shows the UV-visible absorption spectra of different electrodes after being inserted into 0.1 M NaAc / HAc buffer solution (pH 4.0) containing 1 mM H2O2 for 20 minutes. The inset is a photo of the samples after the reaction is completed (ⅰ: ITO; ⅱ: VMSF / ITO; ⅲ: CeO2@VMSF / ITO; ⅳ: PtNPs@VMSF / ITO; v: CeO2-PtNPs@VMSF / ITO).

[0039] Figure 5 To characterize the electrochemical performance of different electrodes in Example 1, Figure 5 (A) ECL intensity plots of different electrodes in 0.01 M PBS (pH = 7.4) containing 100 μM luminol. Figure 5 (B) In the presence of 2.5 mM [Fe(CN)6] 3- / 4- CV curves of different electrodes in 0.1 M KCl solution, Figure 5 (C) in the presence of 2.5 mM [Fe(CN)6] 3- / 4- EIS curves of different electrodes in 0.1 MKCl solution.

[0040] Figure 6 In order to optimize the cyclic voltammetry electrodeposition number of PtNPs nanozyme, the incubation time of IFN-γ antibody and the incubation time of IFN-γ in Example 1, Figure 6 (A) ECL intensity graph of CeO2@VMSF / ITO electrode tested in 0.01 M PBS (pH = 7.4) containing 100 μM luminol after different CV scan cycles in H2PtCl6 solution. Figure 6 Middle (B) shows the BSA / Ab ratio after incubation with 10 μg / mL Ab for different time periods. IFN-γ ECL intensity graph of / CeO2-PtNPs@O-VMSF / ITO electrode measured in 0.01 M PBS (pH 7.4) containing 100 μM luminol. Figure 6 Middle (C) shows IFN-γ / BSA / Ab after incubation with 1 ng / mL IFN-γ for different time periods. IFN-γ ECL intensity graph measured at / CeO2-PtNPs@O-VMSF / ITO electrode in 0.01 M PBS (pH 7.4) containing 100 μM luminol.

[0041] Figure 7 IFN-γ / BSA / Ab in Example 1 IFN-γECL intensity graph and linear relationship graph after incubation of / CeO2-PtNPs@O-VMSF / ITO electrode with different concentrations of IFN-γ, Figure 7 (A) is the ECL intensity map, Figure 7 Middle (B) shows the corresponding linear regression equation, and the error bars represent the standard deviation of three measurements.

[0042] Figure 8 is the BSA / Ab in Example 1 IFN-γ / CeO2-PtNPs@O-VMSF / ITO electrode anti-interference ability and stability test, Figure 8 (A) is BSA / Ab IFN-γ / CeO2-PtNPs@O-VMSF / ITO electrode analysis of different species before ( I 0)After( I ) of the ECL intensity ratio ( I / I 0), the concentration of IFN-γ was 1 ng / mL, the concentrations of IL-6, AFP, TNF-α and IL-1β were 10 ng / mL, the concentrations of AA, Glu, Na + and K + The concentration was 100 μM. Figure 8 Middle (B) shows the ECL signal when five sensing electrodes detect 1 ng / mL IFN-γ in parallel. Figure 8 Middle (C) is BSA / Ab IFN-γ Changes in the ECL intensity ratio of the / CeO2-PtNPs@O-VMSF / ITO electrode before and after the addition of 1 ng / mL IFN-γ after storage for different days. Error bars represent the standard deviation of three measurements. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiment. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Those skilled in the art will make modifications or equivalent substitutions based on understanding the technical solution of the present invention, without departing from the spirit and scope of the technical solution of the present invention, and all should be encompassed within the protection scope of the present invention.

[0044] The raw materials used in the following specific embodiments are all purchased from the market.

[0045] Example 1 The immunoelectrode was prepared by covalently immobilizing the cytokine IFN-γ antibody on the outer surface of a silica nanoporous membrane modified electrode with nanochannel-confined CeO2 / PtNPs composite material. The process of detecting IFN-γ in serum samples by electrochemiluminescence was as follows: (1) Preparation of VMSF / ITO by electrochemically assisted self-assembly (EASA) First, 20 mL of ethanol, 20 mL of NaNO3 (0.1 M, pH = 2.6), and 1.585 g of surfactant cetyltrimethylammonium bromide (CTAB) were added. After the CTAB was completely dissolved and the solution was mixed evenly, 3500 μL of tetraethoxysiloxane (TEOS) was added and the prepared precursor solution was vigorously stirred for 2.5 h.

[0046] The pretreated ITO electrode was placed in the precursor solution using the constant current growth method, and a constant current (j = −0.7 mA / cm 2 ) for 10 s. After the reaction, the electrode was quickly removed, rinsed with plenty of deionized water, and dried with nitrogen. The electrode was then aged in an oven at 120°C overnight to obtain an electrode containing micelles in nanopores, named SM@VMSF / ITO. The SM@VMSF / ITO was immersed in a 0.1 M HCl-ethanol solution with stirring for 5 minutes to remove the micelles, resulting in a VMSF / ITO electrode with open pores.

[0047] (2) Preparation of CeO2@VMSF / ITO CeO2@VMSF / ITO was prepared by confining CeO2 in VMSF using a one-step electrodeposition method. The specific method is as follows: VMSF / ITO was immersed in Ce(NO3)3·6H2O (0.2 M) aqueous solution and a potential of -0.8 V was applied for 15 s to prepare CeO2@VMSF / ITO electrode.

[0048] (3) Preparation of CeO2-PtNPs@VMSF / ITO The PtNPs were deposited in an H2PtCl6 solution (1.93 μM H2PtCl6 in 0.1 M H2SO4). The prepared CeO2@VMSF / ITO electrode was immersed in the electrodeposition solution. Cyclic voltammetry was performed for five cycles in the potential window of -0.4 V to 0.2 V for 120 s. The PtNPs were confined within the nanopores of the VMSF. The resulting electrode was designated CeO2-PtNPs@VMSF / ITO.

[0049] (4) Preparation of immunosensor The antibody and VMSF layers were covalently crosslinked using γ-glycidyloxypropyltrimethylsilane (GPTMS) as a crosslinker. The specific experimental steps are as follows: a CeO2-PtNPs@VMSF / ITO electrode was immersed in a 2.26 mM GPTMS ethanol solution at room temperature for 1 hour. GPTMS was modified on the VMSF surface via silanization reaction. The electrode was then washed with deionized water to obtain a CeO2-PtNPs@O-VMSF / ITO electrode.

[0050] Take IFN-γ antibody (50 μL, 10 μg mL -1 ) was drop-coated on the surface of the CeO2-PtNPs@O-VMSF / ITO electrode and incubated at 4°C for 60 min. The electrode surface was carefully rinsed with PBS (0.01 M, pH = 7.4) to remove unbound antibodies. The resulting electrode was incubated with BSA solution at room temperature for 10 min to block nonspecific binding sites, and the electrode was rinsed with PBS (0.01 M, pH = 7.4) to remove unbound BSA. It was then blown dry with N2 to obtain the final immunosensor, which was recorded as BSA / Ab. IFN-γ / CeO2-PtNPs@O-VMSF / ITO and stored at 4 °C for subsequent use.

[0051] (5) Electrochemiluminescence detection of IFN-γ To detect IFN-γ, BSA / Ab IFN-γ The / CeO2-PtNPs@O-VMSF / ITO electrode was incubated with 50 μL, 1 ng / mL IFN-γ at 4°C for 60 min, and the electrode was slowly rinsed with PBS (0.01 M, pH = 7.4) solution to wash away the unbound IFN-γ. The resulting electrode was recorded as IFN-γ / BSA / Ab IFN-γ / CeO2-PtNPs@O-VMSF / ITO.

[0052] The resulting electrode was subjected to ECL detection using a 100 μM luminol-containing electrolyte solution in PBS (0.01 M, pH 7.4). The ECL process was triggered by continuous CV scans over a potential range of −1 to 0.8 V at a scan rate of 100 mV / s.

[0053] Electron microscopy of the ITO electrode modified with silica nanoporous film (VMSF / ITO) prepared by electrochemically assisted self-assembly Figure 1 shown.

[0054] Figure 1The SEM characterization results of (A) show that the VMSF / ITO modified electrode presents a clear three-layer structure, consisting of a base glass layer, an ITO conductive layer, and a 95 nm thick VMSF functional layer on the surface, with distinct interface transition regions. Figure 1 The TEM characterization results in (B) show that the VMSF surface presents a typical hexagonal mesoporous structure with a uniform pore size distribution (2 ~ 3 nm). The film remains intact as a whole without cracks. Figure 1 The cross-sectional TEM image in (C) further shows a long-range ordered array of nanochannels with a longitudinal length of approximately 94 nm and good thickness uniformity, indicating that the material has excellent mechanical stability.

[0055] Figure 2 XPS characterization of VMSF / ITO, CeO2@VMSF / ITO and CeO2-PtNPs@VMSF / ITO. Figure 2 (A) is the XPS total spectrum of VMSF / ITO, CeO2@VMSF / ITO and CeO2-PtNPs@VMSF / ITO. After electrodeposition of CeO2 and PtNPs, characteristic peaks of Ce 3d and Pt 4f energy levels appeared in the CeO2-PtNPs@VMSF / ITO electrode. Figure 2 Middle (B)~ Figure 2 (D) are high-resolution spectra of O 1s, Ce 3d and Pt 4f in CeO2-PtNPs@VMSF / ITO electrode. The peak at 531.8 eV in the O 1s spectrum is the characteristic peak of Si-O, the peak at 530.1 eV is the characteristic peak of Ce-O, and the peak at 533.2 eV is the characteristic peak of O v Characteristic peaks, including O v The appearance of the signal proves that there are oxygen defects on the surface of CeO2 nanozymes, which proves the possible existence of Ce 3+ .

[0056] In the Ce 3d high-resolution spectrum, the three characteristic peaks at 916.2 eV, 903.7 eV and 900.2 eV (denoted as u1, u2, and u3) are respectively attributed to Ce 4+ 3d 3 / 2 energy level, and the three characteristic peaks at 897.7 eV, 885.2 eV and 881.6 eV (denoted as v1, v2, v3) are attributed to Ce 4+ 3d 5 / 2 It is worth noting that the high-resolution spectrum also has peaks at 906.7 eV and 888.7 eV (denoted as w1 and w2), which are Ce 3+ It is speculated that the mesoporous nanochannels restrict the growth of CeO2 nanozymes, making it easier for CeO2 to appear on the surface. 3+, thus forming oxygen vacancies (O v In the Pt 4f high-resolution spectrum, the two characteristic peaks at 73.1 eV and 73.8 eV prove that metallic Pt 0 , thus confirming the successful confinement of PtNPs.

[0057] Figure 3 The electrodeposition of CeO2, PtNPs and CeO2-PtNPs on ITO electrode and VMSF / ITO electrode were compared. Figure 3 (A) shows the results of continuous ECL measurements of the ITO electrode and after direct electroplating of CeO2, PtNPs, and CeO2-PtNPs on the ITO electrode. The experimental steps are the same as those in Example 1, except that the ITO electrode was not modified with VMSF and step (1) was omitted. Compared with the ITO electrode, the CeO2@ITO, PtNPs@ITO, and CeO2-PtNPs@ITO electrodes showed higher ECL signals during the initial scan. However, as the scan time increased, the ECL signal decreased significantly and eventually became almost the same as that of the ITO electrode. This may be because when the nanozyme is directly deposited on the surface of the ITO electrode, the nanocatalyst cannot exist stably. During the electrode scanning process, the peeling of the nanomaterial leads to a decrease in ECL performance.

[0058] As a control, Figure 3 (B) shows the sequential ECL measurements of a VMSF / ITO electrode after electrodeposition of CeO2, PtNPs, and CeO2-PtNPs on the VMSF / ITO electrode. It can be seen that the ECL signal of the electrode after confining the CeO2-PtNPs composite nanozyme is significantly higher than that of the single nanozyme and exhibits good stability. This result indicates that CeO2 and PtNPs have a coordinated catalytic effect. Furthermore, the ECL signal measured on PtNPs@VMSF / ITO is significantly lower than that on PtNPs@ITO, indicating that the amount of PtNPs deposited in the ultrasmall nanochannels is relatively small. However, the ECL signal on the CeO2-PtNPs@VMSF / ITO electrode is significantly higher than that measured on the CeO2-PtNPs@ITO electrode. Therefore, the coordinated effect of the nanomaterials confined in the nanochannels enables CeO2-PtNPs to exhibit higher catalytic activity.

[0059] Figure 4 The electrochemical behavior and ECL intensity changes of CeO2-PtNPs@VMSF / ITO electrode under different atmospheres were discussed. Figure 4 The electrochemical behavior and ECL intensity changes of CeO2-PtNPs@VMSF / ITO electrode in Example 1 under different atmospheres are shown. Figure 4 (A) is the CV curve of the electrode under different atmospheres. Figure 4Middle (B) is the ECL signal of the electrode under different atmospheres. Figure 4 (C) is the ECL potential diagram of the electrode in 0.01 M PBS (pH = 7.4) containing 100 μM luminol when the starting scan potential is 0 V and the initial scan direction is positive (left) and negative (right). I and I 0 represents the ECL signal in the presence and absence of free radical scavengers, respectively. Figure 4 (D) shows the ECL response of the electrode in the luminol-O2 system after adding different free radical scavengers. Figure 4 Middle (E) shows the UV-visible absorption spectra of different electrodes after being inserted into 0.1 M NaAc / HAc buffer solution (pH 4.0) containing 0.25 mM TMB and 1 mM H2O2 for 20 min. Figure 4 Middle (F) shows the UV-visible absorption spectra of different electrodes after being inserted into 0.1 M NaAc / HAc buffer solution (pH 4.0) containing 1 mM H2O2 for 20 min. The inset is a photo of the samples after the reaction is completed (ⅰ: ITO; ⅱ: VMSF / ITO; ⅲ: CeO2@VMSF / ITO; ⅳ: PtNPs@VMSF / ITO; v: CeO2-PtNPs@VMSF / ITO).

[0060] like Figure 4 As shown in (A), as the dissolved oxygen content in the solution increases, the oxygen reduction peak current gradually increases, and the corresponding ECL signal also increases synchronously ( Figure 4 (B)). However, under conditions of near-saturation with N₂, no significant electrochemiluminescence was observed, indicating that without O₂ generating ROS, luminol struggles to reach an excited state and thus fails to emit light. By altering the electrochemical process that excites ECL and the order of luminol oxidation and dissolved oxygen reduction, the mechanism of enhanced ECL in the luminol-DO system using CeO₂-PtNPs composite nanozymes was further investigated. The initial electrochemical scan began at 0 V, with both anodic and cathodic scanning modes.

[0061] Figure 4 (C) shows the ECL signals generated when the CeO2-PtNPs@VMSF / ITO electrode is scanned towards the anode and cathode respectively at a starting potential of 0 V. It can be seen that when the CeO2-PtNPs@VMSF / ITO electrode is scanned towards the anode, the oxidation reaction of luminol first occurs to generate L •- , followed by the reduction reaction of O2 to produce O2 •-In the scanning cycle with the initial direction being the anodic direction, no obvious ECL signal was observed, which may be because L •- The number is small and the life span is short, and it fails to •- When the CeO2-PtNPs@VMSF / ITO electrode is scanned toward the cathode at a starting potential of 0 V, the first generated O2 •- Can survive to L •- It generates and reacts with it, producing obvious anodic luminescence. This indicates that O2 •- The lifespan is L •- The results also demonstrate that CeO2-PtNPs composite nanozymes can catalyze the reduction of O2, generating more ROS, thereby significantly enhancing the ECL intensity. Free radical capture confirmed the main reactive oxygen species generated by CeO2-PtNPs composite nanozymes during the ECL process.

[0062] like Figure 4 As shown in (D), when the hydroxyl radical (OH•) scavenger isopropyl alcohol (IPA) was added, the ECL signal measured on the electrode did not change significantly. When the H2O2 scavenger catalase (CAT) was added, the ECL signal measured on the electrode decreased by about 40%. •- After the capture agent benzoquinone (BQ), the ECL signal was almost completely quenched. This proves that the main ROS generated during the ECL process of the luminol-DO system catalyzed by CeO2-PtNPs is O2 •- and H2O2. ROS is used to oxidize colorless TMB to generate blue oxidized TMB with characteristic absorption at 650 nm. + , the enzyme-mimicking activity of confined CeO2-PtNPs was investigated.

[0063] like Figure 4 As shown in (E), under the same reaction time and with H2O2 as substrate, the absorbance value of the electrode solution after confining the two single nanozymes increased, indicating that both CeO2 and PtNPs have good peroxidase activity, but the electrode confining CeO2-PtNPs composite nanozyme showed better POD activity. Figure 4 In (F), the enzymatic reaction using dissolved oxygen as a substrate shows that both the electrodes containing a single nanozyme or a composite nanozyme exhibit good oxidase (OXD) activity. Therefore, the composite nanozyme synthesized by electrochemical deposition not only has POD activity but also OXD activity.

[0064] Electrochemiluminescence (ECL), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to characterize the BSA / Ab IFN-γ The construction process of / CeO2-PtNPs@VMSF / ITO immunosensor was characterized. Figure 5 To characterize the electrochemical performance of different electrodes in Example 1, Figure 5 (A) ECL intensity plots of different electrodes in 0.01 M PBS (pH = 7.4) containing 100 μM luminol. Figure 5 (B) In the presence of 2.5 mM [Fe(CN)6] 3- / 4- CV curves of different electrodes in 0.1 M KCl solution, Figure 5 (C) in the presence of 2.5 mM [Fe(CN)6] 3- / 4- EIS curves of different electrodes in 0.1 M KCl solution.

[0065] like Figure 5 As shown in (A), due to the catalytic activity of CeO2-PtNPs composite nanozymes, confining CeO2-PtNPs in nanochannels significantly enhanced the ECL signal. GPTMS modified the CeO2-PtNPs@VMSF / ITO electrode to functionalize its outer surface, increasing the steric hindrance, resulting in the diffusion of luminol molecules being hindered and the ECL signal slightly decreasing. After the antibody was modified on the surface of CeO2-PtNPs@O-VMSF / ITO electrode, the amino group of the antibody formed an amide bond with the alkoxy group, further hindering the diffusion of luminol molecules, and the ECL signal further decreased. Subsequently, BSA / Ab IFN-γ After incubation of the / CeO2-PtNPs@VMSF / ITO electrode with IFN-γ, a huge protein immune complex was formed on the electrode surface due to the specific binding between the antigen and the antibody, which blocked the nanopores and further hindered the diffusion of luminol to the electrode surface, causing the ECL signal to continue to decrease. 3− / 4− As a signal probe, the construction process of the immunosensor was verified by CV and EIS.

[0066] like Figure 5 Middle (B) and Figure 5 As shown in (C), the CeO2-PtNPs@VMSF / ITO electrode is highly efficient in mass transfer due to the open silica nanochannels. 3- / 4- The solution showed obvious redox peaks, large anodic peak current and relatively small charge transfer resistance (Rct). With the gradual modification, [Fe(CN)6] 3- / 4- The diffusion of the nanostructured carbon is hindered, and the curve shows a trend similar to that of ECL.

[0067] In order to obtain the best detection performance, the experimental conditions were optimized, including the number of cyclic voltammetry electrodeposition cycles of PtNPs nanozymes, the incubation time of IFN-γ antibody, and the incubation time of IFN-γ. Figure 6 As shown, Figure 6 (A) ECL intensity graph of CeO2@VMSF / ITO electrode tested in 0.01 M PBS (pH = 7.4) containing 100 μM luminol after different CV scan cycles in H2PtCl6 solution. Figure 6 Middle (B) shows the BSA / Ab ratio after incubation with 10 μg / mL Ab for different time periods. IFN-γ ECL intensity graph of / CeO2-PtNPs@O-VMSF / ITO electrode measured in 0.01 M PBS (pH 7.4) containing 100 μM luminol, Figure 6 Middle (C) shows IFN-γ / BSA / Ab after incubation with 1 ng / mL IFN-γ for different time periods. IFN-γ ECL intensity graph measured at / CeO2-PtNPs@O-VMSF / ITO electrode in 0.01 M PBS (pH 7.4) containing 100 μM luminol.

[0068] Figure 6 Figure (A) shows the ECL intensity of a CeO2@VMSF / ITO electrode after various CV scan cycles in H2PtCl6 solution. The electrode's ECL intensity initially increases and then decreases with increasing deposition cycles, reaching a maximum at 5 cycles. This is due to the following reasons: When the electrodeposition cycle is too short, the CeO2-PtNPs@VMSF / ITO electrode has fewer active sites on its surface, resulting in insufficient reactive oxygen species (ROS) generation and a lack of strong ECL emission. When the electrodeposition time is too long, the excess CeO2-PtNPs nanozymes clog the nanochannels, hindering ion transport and electrochemical interface dynamics. Considering experimental cost and time efficiency, the antibody incubation time was optimized during the preparation of the immunosensor interface.

[0069] like Figure 6 As shown in (B), the ECL signal gradually decreased with the increase of antibody incubation time. When the antibody incubation time was 60 minutes, the ECL signal was stable. Further increase in the antibody incubation time had little effect on the ECL signal, indicating that the antibody fixation had reached saturation at 60 minutes. Therefore, a 60-minute incubation time was selected for subsequent studies. At the same time, the incubation time of IFN-γ was also optimized, as shown in Figure 5. Figure 6 As shown in (C), the ECL signal stabilized after 60 minutes of IFN-γ incubation, indicating that IFN-γ reached saturation at 60 minutes.

[0070] Under optimal conditions, the constructed immunosensor was incubated with different concentrations of IFN-γ, and the ECL signal of the electrode was measured in a luminol-DO system. Figure 7 IFN-γ / BSA / Ab in Example 1 IFN-γ ECL intensity and linear relationship diagram of the / CeO2-PtNPs@O-VMSF / ITO electrode after incubation with different concentrations (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) of IFN-γ in 0.01 M PBS (pH 7.4) containing 100 μM luminol. Figure 7 (A) is the ECL intensity map, Figure 7 Middle (B) shows the corresponding linear regression equation, and the error bars represent the standard deviation of three measurements.

[0071] like Figure 7 Middle (A) and Figure 7 As shown in (B), it can be observed that the ECL signal decreases with increasing IFN-γ concentration. This is attributed to the formation of immune complexes on the electrode surface, which increases the interfacial resistance of the electrode and reduces the diffusion of luminol. -1 to 10 ng mL -1 In the concentration range, the ECL intensity (I ECL ) is linearly related to the logarithm of IFN-γ concentration (logC), and the corresponding linear regression equation is: I ECL = (-821.3 ± 26) Log C IFN-γ (pg·mL -1 ) + (8488 ±57), R 2 The value was 0.994. Based on a signal-to-noise ratio of 3 (S / N = 3), the limit of detection (LOD) was calculated to be 4.2 fg mL -1 This indicates that the prepared immunosensor can be used in the field of clinical diagnosis to detect the level of IFN-γ in human blood samples.

[0072] In order to evaluate the selectivity and anti-interference of the sensor, cytokines IL-6, TNF-α and IL-1β, tumor marker AFP, and serum K + 、Na + , glucose and ascorbic acid as interferors. Figure 8 is the BSA / Ab in Example 1 IFN-γ / CeO2-PtNPs@O-VMSF / ITO electrode anti-interference ability and stability test, Figure 8 (A) is BSA / Ab IFN-γ / CeO2-PtNPs@O-VMSF / ITO electrode analysis of different species before ( I 0)After( I ) of the ECL intensity ratio ( I / I 0). The concentration of IFN-γ was 1 ng / mL, the concentrations of IL-6, AFP, TNF-α and IL-1β were 10 ng / mL, and the concentrations of AA, Glu, Na + and K + The concentration is 100 μM, Figure 8 Middle (B) shows the ECL signal when five sensing electrodes detect 1 ng / mL IFN-γ in parallel. Figure 8 Middle (C) is BSA / Ab IFN-γ Changes in the ECL intensity ratio of the / CeO2-PtNPs@O-VMSF / ITO electrode after storage for different days before and after the addition of 1 ng / mL IFN-γ. Error bars represent the standard deviation of three measurements.

[0073] like Figure 8 As shown in (A), it is clear that the immunosensor will show a significant decrease in ECL signal only when the target analyte is present, while the effect of other substances on the decrease in ECL signal is negligible, which shows that the prepared sensor has selectivity and anti-interference properties. In addition, the reproducibility of the sensor was explored. Figure 8 As shown in (B), five electrodes were prepared under the same conditions for the detection of IFN-γ, and the relative standard deviation (RSD) was only 2.7%, confirming that the sensor has good reproducibility. Figure 8 Middle (C) shows the stability of the prepared immunosensor stored at 4°C. After 10 days of storage, the ECL intensity of the immunosensor did not change significantly, and the RSD value within 9 days was 3.5%, indicating that the sensor has good long-term stability.

Claims

1. An immunosensor for electrochemiluminescence detection of cytokine interferon-γ, characterized in that: The immunosensor includes a three-electrode system of an immunoelectrode, a reference electrode, and a counter electrode; The immunoelectrode is a VMSF-modified electrode containing ceria nanozymes and platinum nanoparticles, which is obtained by covalently fixing IFN-γ antibodies and blocking nonspecific sites. The immunosensor uses a solution containing luminol as an electrolyte during detection.

2. The electrochemiluminescence immunosensor for detecting cytokine interferon-γ according to claim 1, characterized in that: The immunoelectrode preparation specifically includes the following steps: Step 1: immerse the VMSF modified electrode in a cerium salt solution and electrodeposit to obtain a confined CeO2 electrode; Step 2, immersing the confined CeO2 electrode in a H2PtCl6 solution for electrodeposition to obtain a confined CeO2-PtNPs electrode; Step 3, immersing the confined CeO2-PtNPs electrode in an organosiloxane solution containing epoxy groups and allowing it to react to obtain an epoxy VMSF functionalized electrode; Step 4: immerse the electrode obtained in step 3 in a buffer solution containing IFN-γ antibody and incubate it, and then immerse it in a buffer solution containing BSA and incubate it to obtain the immunoelectrode.

3. The immunosensor for detecting cytokine interferon-γ by electrochemiluminescence according to claim 2, characterized in that: The cerium salt includes any one or more of Ce(NO3)3·6H2O, Ce(NH4)2(NO3), and CeCl3; And / or, the organosiloxane includes any one or more of γ-glycidyloxypropyltrimethylsilane, 3-(2,3-epoxypropyloxy)propyltrimethoxysilane, and 2-(3,4-epoxycyclohexyl)ethyltriethoxysilane; And / or, the solvent used in the cerium salt solution is water; the solvent used in the H2PtCl6 solution is water or sulfuric acid; the solvent used in the organosiloxane solution includes any one or more of acetone, ethanol, methanol, and cyclohexane; And / or, the buffer solution includes a sodium chloride aqueous solution, a sodium sulfate aqueous solution, a potassium chloride aqueous solution or a phosphate buffer solution; the solute concentration in the buffer solution is 0.005 to 0.5 mol / L, and the pH is 6 to 8.

4. The immunosensor for detecting cytokine interferon-γ by electrochemiluminescence according to claim 2, characterized in that: The molar concentration of the cerium salt in the cerium salt solution in step 1 is 0.1 to 0.2 M, the electrodeposition time is 10 to 20 s, and the electrodeposition voltage is -0.7 to -0.8 V; And / or, the H2PtCl6 concentration in the H2PtCl6 solution in step 2 is 0.1-1.0 μM, the electrodeposition time is 100 ~120 s, and the electrodeposition voltage is -0.4 V ~ 0.2 V.

5. The immunosensor for detecting cytokine interferon-γ by electrochemiluminescence according to claim 2, characterized in that: In step 3, the molar concentration of the organosiloxane in the organosiloxane solution is 2.0 mM to 3.0 mM, and the reaction is allowed to stand for 30 to 60 minutes at a temperature of 20 to 40°C. And / or, in step 4, the electrode is incubated in the buffer solution containing IFN-γ antibody at 0 to 5° C. for 1 to 2 hours; and incubated in the buffer solution containing BSA at room temperature for 5 to 30 minutes; And / or, in step 4, the concentration of the IFN-γ antibody in the buffer solution containing the IFN-γ antibody is 1 to 20 μg / mL.

6. The electrochemiluminescence immunosensor for detecting cytokine interferon-γ according to claim 1, characterized in that: The molar concentration of luminol in the detection solution is 5 to 300 μmol / L.

7. The method for preparing the electrochemiluminescence immunosensor for detecting cytokine interferon-γ according to any one of claims 1 to 6, characterized in that: Including steps: Step 1: immerse the VMSF modified electrode in a cerium salt solution and electrodeposit to obtain a confined CeO2 electrode; Step 2, immersing the confined CeO2 electrode in a H2PtCl6 solution for electrodeposition to obtain a confined CeO2-PtNPs electrode; Step 3, immersing the confined CeO2-PtNPs electrode in an organosiloxane solution containing epoxy groups and allowing it to react to obtain an epoxy VMSF functionalized electrode; Step 4, immersing the electrode obtained in step 3 in a buffer solution containing IFN-γ antibody and incubating, and then immersing it in a buffer solution containing BSA and incubating to obtain the immunoelectrode; Step 5: forming a three-electrode system with the immunoelectrode, the counter electrode and the reference electrode to obtain the immunosensor, wherein the immunosensor uses a solution containing luminol as an electrolyte during detection.

8. Use of the electrochemiluminescence immunosensor for detecting cytokine interferon-γ according to any one of claims 1 to 6 in detecting cytokine interferon-γ for non-diagnostic or non-therapeutic purposes.

9. A method for detecting cytokine interferon-γ by electrochemiluminescence, characterized in that: The immunoelectrode according to any one of claims 1 to 6 is incubated with a test solution and then rinsed to obtain a test electrode; the test electrode is used as a working electrode, and a three-electrode system is formed with a reference electrode and a counter electrode, and an electrolyte solution containing luminol is used as a detection solution for electrochemiluminescence signal detection.

10. The method for detecting cytokine interferon-γ by electrochemiluminescence according to claim 9, characterized in that: The concentration of cytokine interferon γ in the test solution is below 100 ng / mL; And / or, the incubation is at 0-5°C for 5-120 min; And / or, the test solution includes one or more interfering compounds selected from glucose, inorganic salts, cytokines, and tumor markers; and the total molar concentration of the interfering compounds is below 0.5M.

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