Electrochemiluminescence immunosensor for detecting interleukin-1 beta, preparation method and application thereof
By constructing a Co3O4/NGQDs composite nanozyme in an SNF nanoreactor, the problem of high-sensitivity quantitative detection of interleukin-1β under neutral conditions in existing electrochemiluminescence detection methods has been solved, achieving efficient and stable detection results, and is suitable for direct detection of biological samples.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HANGZHOU FIRST PEOPLES HOSPITAL
- Filing Date
- 2026-04-16
- Publication Date
- 2026-05-12
AI Technical Summary
Existing electrochemiluminescence detection methods are difficult to achieve highly sensitive and accurate quantitative detection of interleukin-1β under neutral conditions, and the introduction of exogenous hydrogen peroxide increases the complexity of the system and background noise.
A Co3O4/NGQDs composite nanozyme was constructed in an ordered mesoporous silica thin film (SNF) nanoreactor. The confinement effect of SNF enabled Co3O4 and NGQDs to be tightly coupled, forming a synergistic catalytic interface. Combined with a luminol/dissolved oxygen system, the use of exogenous H2O2 was avoided, and it was adapted to neutral physiological pH conditions.
It significantly enhances the catalytic efficiency of the luminol/dissolved oxygen system, achieves high-sensitivity detection under neutral conditions, has a wide detection range, low detection limit, adapts to the physiological environment of biological samples, requires no sample pH pre-adjustment, has high detection stability, and is suitable for large-scale applications.
Smart Images
Figure CN122017225A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of electrochemiluminescence sensing technology, and in particular to an electrochemiluminescence immunosensor for detecting interleukin-1β, its preparation method, and its application. Background Technology
[0002] Interleukin-1β (IL-1β) is a core pro-inflammatory cytokine. In obstetrics, its dynamic changes are a key biomarker for assessing the immune status and inflammatory balance at the maternal-fetal interface, and are of great significance in evaluating major pregnancy complications. Currently, widely used methods for detecting IL-1β include enzyme-linked immunosorbent assay (ELISA), colorimetric methods, and chromatographic methods. However, these methods involve complex sample pretreatment and require expensive, large-scale instruments.
[0003] Electrochemiluminescence (ECL) analysis is an analytical method that excites luminescent reactions through electrochemical means. It boasts significant advantages such as low background signal, ease of operation, wide dynamic range, and strong spatiotemporal controllability, making it a commonly used analytical tool in fields such as biosensing, environmental monitoring, and clinical diagnostics. Among ECL analysis techniques, the luminol / co-reactant system has attracted widespread attention from researchers due to its low excitation potential and broad analytical applications. However, this system is still limited by low ECL quantum yield and insufficient signal intensity; therefore, improving the ECL intensity of the luminol / co-reactant system using conventional electrodes is crucial.
[0004] Chinese patent application CN119086677A discloses a nanozyme-sensitized electrochemiluminescence immunoassay biosensor, its construction method, and its application. The method involves modifying the electrode with a silica nanoporous membrane containing a Co3O4 nanozyme confined in a nanochannel. The Co3O4 nanozyme performs dual electrocatalysis on luminol and hydrogen peroxide to enhance the signal of the luminol / hydrogen peroxide system, where hydrogen peroxide is an exogenous co-reactant. However, this approach has the following limitations: First, the system relies on the external addition of unstable hydrogen peroxide, increasing system complexity and background noise; second, the catalytic efficiency is limited by the activity of the single nanozyme; third, the system typically operates under alkaline conditions, which is incompatible with the neutral physiological environment of biological samples, requiring additional pH adjustment of the sample. Furthermore, while the luminol / dissolved oxygen system using an endogenous co-reactant avoids the introduction of exogenous hydrogen peroxide, this system exhibits weak signal and low detection sensitivity under neutral conditions. In summary, existing electrochemiluminescence detection systems are insufficient for achieving simple, highly sensitive, and accurate quantitative detection of interleukin-1β. Summary of the Invention
[0005] To address the aforementioned technical problems, this invention provides an electrochemiluminescent immunosensor for detecting interleukin-1β, which can significantly enhance the signal of the luminol / dissolved oxygen system and achieve highly sensitive and accurate quantitative detection of interleukin-1β under neutral conditions.
[0006] The first specific technical solution of the present invention is: an electrochemiluminescent immunosensor for detecting interleukin-1β, comprising a working electrode, a reference electrode and a counter electrode; The working electrode includes a base electrode and an ordered mesoporous silica film (SNF) modified on the surface of the base electrode. The SNF has a nanopore loaded with a composite nanozyme containing cobalt tetroxide (Co3O4) nanoparticles and nitrogen-doped graphene quantum dots (NGQDs), and the outer surface of the SNF is modified with epoxy groups, and an anti-interleukin-1β antibody is covalently fixed through the epoxy groups. The detection system of the electrochemiluminescence immunosensor is a luminol / dissolved oxygen system.
[0007] This invention constructs a Co3O4 / NGQDs composite nanozyme in a SNF nanoreactor. Utilizing the confinement effect of SNF, Co3O4 and NGQDs form a tightly coupled synergistic catalytic interface, resulting in a significant synergistic catalytic effect. Compared to single Co3O4 nanozymes or NGQDs components, this significantly improves the catalytic efficiency of the luminol / dissolved oxygen system and enhances the luminol / dissolved oxygen system signal. This allows luminol to achieve efficient electrochemiluminescence under neutral physiological pH conditions. Combined with an immunorecognition interface, it can be applied to the highly sensitive detection of IL-1β. The sensor constructed in this way is directly compatible with the physiological environment of biological samples such as serum, eliminating the need for pH pre-adjustment of the test sample and simplifying the detection operation. Simultaneously, using dissolved oxygen in the environment as the sole co-reactant makes the system more stable and environmentally friendly, avoiding problems such as easy decomposition of exogenous H2O2, background noise, and potential interference with biomolecules. It achieves highly sensitive and accurate quantitative detection of interleukin-1β under neutral conditions, showing significant practical application potential for early warning of pregnancy complications.
[0008] Optionally, the spacing between the Co3O4 nanoparticles and adjacent NGQDs in the nanopores is no greater than 3 nm.
[0009] Optionally, the pH value of the detection system is 6.8~7.6.
[0010] Optionally, the substrate electrode includes any one of indium tin oxide electrode, glassy carbon electrode, fluorine-doped tin oxide electrode, gold electrode, screen-printed electrode, graphite electrode, and carbon fiber electrode.
[0011] Optionally, the reference electrode includes one or more of Ag / AgCl electrode (saturated KCl), saturated calomel electrode, and Hg / HgO electrode, and the counter electrode includes one or more of platinum wire and platinum sheet.
[0012] The second specific technical solution of the present invention is: a method for preparing an electrochemiluminescent immunosensor for detecting interleukin-1β, comprising the following steps: (1) Modify SNF on the surface of the substrate electrode to obtain an SNF-modified electrode, immerse it in a silane solution containing epoxy groups to react, and obtain an O-SNF-modified electrode with epoxy groups on the outer surface. (2) The O-SNF modified electrode was immersed in a solution containing CoSO4 for electrodeposition to obtain Co3O4@O-SNF modified electrode; (3) The Co3O4@O-SNF modified electrode was immersed in a solution containing NGQDs for electrophoresis treatment to obtain the NGQDs / Co3O4@O-SNF modified electrode; (4) The NGQDs / Co3O4@O-SNF modified electrode was incubated with a buffer solution containing anti-interleukin-1β antibody, and the non-specific sites were blocked with bovine serum albumin (BSA) solution to obtain the working electrode.
[0013] In the above preparation method, the substrate electrode surface is modified with SNF. SNF has a uniform and ordered nanoporous structure, which can provide a stable confined space for subsequent loading of nanocomponents. The SNF is then immersed in a silane solution containing epoxy groups for reaction, and epoxy active groups are modified on the outer surface of the SNF to connect to the antibody. The O-SNF-modified electrode is then immersed in a solution containing CoSO4 for electrodeposition, allowing the cobalt-based precursor to be generated in situ within the SNF channels and transformed into Co3O4 nanoparticles. This achieves high dispersion and stable loading of Co3O4 within the channels. Then, C... The o3O4@O-SNF modified electrode is immersed in a solution containing NGQDs for electrophoresis. Under the drive of an electric field, the NGQDs enter the SNF nanopores and bind tightly with the Co3O4 nanoparticles inside the pores to form a Co3O4 / NGQDs composite nanozyme. The two produce a significant synergistic catalytic effect, which greatly improves the catalytic efficiency of the luminol / dissolved oxygen system and significantly enhances the ECL signal. The change in the electrode ECL signal caused by the binding of the recognition antibody to IL-1β ultimately enables the quantitative detection of IL-1β.
[0014] Optionally, in step (1), the concentration of the silane containing the epoxy group in the silane solution is 2.0 ~ 3.0 mmol / Lol / L.
[0015] Optionally, in step (1), the reaction time is 30-60 min and the reaction temperature is 20-40℃.
[0016] Optionally, in step (1), the SNF is modified on the substrate electrode surface using the Stöber solution growth method.
[0017] Optionally, in step (2), the concentration of CoSO4 in the CoSO4-containing solution is 0.02~0.2 mol / L; the electrodeposition voltage is +1.0 ~ +2.0 V, and the deposition time is 1 ~ 20 s.
[0018] Optionally, in step (3), the electrophoresis treatment potential is +0.4 ~ +1.0 V, and the electrophoresis time is 30 ~ 180 s.
[0019] Optionally, in step (3), the NGQDs are obtained by hydrothermal reaction of 1-aminopyrene and ammonia. Specifically, 1-aminopyrene is added to ammonia, the mixture is placed in a polytetrafluoroethylene liner, and the mixture is reacted under high pressure at 150-250°C for 6-10 hours in a reactor. The product is filtered, dialyzed, and freeze-dried to obtain solid NGQDs. The above solid NGQDs powder is then dispersed in deionized water and ultrasonically treated to ensure uniform dispersion, thus obtaining a solution containing NGQDs.
[0020] Optionally, in step (4), the incubation temperature is 0~5℃ and the time is 0.5~3 h.
[0021] Optionally, in step (4), the concentration of interleukin-1β antibody in the solution containing anti-interleukin-1β antibody is 1~20 μg / mL, and the buffer solution is an aqueous solution of sodium chloride, an aqueous solution of sodium sulfate, an aqueous solution of potassium chloride, or a phosphate buffer solution; the solute concentration in the buffer solution is 0.01~0.1 mol / L, and the pH is 6.5~7.5.
[0022] The third specific technical solution of the present invention is: the application of an electrochemiluminescence immunosensor in the detection of interleukin-1β for non-diagnostic purposes, wherein the electrochemiluminescence immunosensor is the aforementioned electrochemiluminescence immunosensor, comprising the following steps: incubating the working electrode of the electrochemiluminescence immunosensor with a sample solution containing interleukin-1β to form an immune complex; placing the incubated working electrode together with a reference electrode and a counter electrode in a neutral buffer solution containing luminol, using dissolved oxygen as a co-reactant, applying a scanning voltage, and quantitatively analyzing interleukin-1β by detecting the degree of reduction in the electrochemiluminescence signal.
[0023] Optionally, the concentration of interleukin-1β in the test solution containing interleukin-1β is 100 fg / mL to 100 ng / mL. In actual testing, due to the high concentration of interleukin-1β in the serum sample or the presence of many interfering substances, the sample can be diluted with an electrolyte solution, such as by a 1-100-fold dilution, to reduce the concentration of interleukin-1β. The test solution may also include one or more interfering proteins such as tumor necrosis factor-α, interleukin-6, and neutrophil gelatinase-associated lipotransferase. Because the immunosensor in this invention is covalently grafted with a specific antibody of the target protein, it exhibits specific selection for the target protein, is less susceptible to interference from other proteins, has high detection stability, and a wide range of applications.
[0024] Optionally, the pH value of the test solution containing interleukin-1β is 6 to 8, preferably 6.5 to 7.5.
[0025] The pH range of the test solution can promote the deprotonation of luminol, resulting in high electrochemiluminescence intensity and sensitivity, and high detection stability.
[0026] Optionally, the concentration of luminol in the luminol / dissolved oxygen system detection solution is 10~200 μmol / L.
[0027] Within this concentration range, the ECL signal will be kept within a suitable absolute value range, resulting in higher sensitivity and cost-effectiveness of this detection technology.
[0028] Compared with the prior art, the present invention has at least the following advantages: (1) The electrochemiluminescence immunosensor of the present invention constructs a Co3O4 / NGQDs composite nanozyme in an SNF nanoreactor. The two are tightly coupled in the confined space, producing a significant synergistic catalytic effect, which greatly enhances the ECL signal of the luminol / dissolved oxygen system. Under pH 7.4 conditions, the ECL signal of this system is more than 2.5 times higher than that of the system with Co3O4 confined alone in the same SNF, and more than 10 times higher than that of the system with NGQDs confined alone. (2) The electrochemiluminescence immunosensor of the present invention shows a linear relationship between the logarithm of the detection concentration of interleukin-1β in the range of 100 fg / mL to 100 ng / mL, and the detection limit is 28 fg / mL. This detection method has a wide linear range, high detection sensitivity and low detection limit, and can achieve the detection of IL-1β with high sensitivity and a wide linear range, which can meet the needs of early IL-1β trace detection in pregnancy complications. (3) The electrochemiluminescence immunosensor of the present invention uses dissolved oxygen in the environment as the only co-reactant, making the system more stable and environmentally friendly. It avoids problems such as easy decomposition of exogenous H2O2, easy generation of background noise and potential interference to biomolecules, making the detection system more stable and more environmentally friendly. (4) The sensing system constructed in this invention can achieve efficient electrochemiluminescence under neutral conditions and is highly compatible with the physiological pH environment of biological samples such as blood, amniotic fluid, and saliva. It does not require pH pre-adjustment of the sample and can be directly used for the detection of diluted real samples, which improves the convenience and accuracy of detection and is suitable for large-scale applications. (5) In this invention, the immune sensor is covalently grafted with a specific antibody of the protein to be detected. Therefore, it has specific selection of the target protein and is not easily affected by other interfering proteins in the test solution. It has strong detection targeting and high stability. Attached Figure Description
[0029] Figure 1 This is the ultraviolet spectrum of the nitrogen-doped graphene quantum dots prepared in Example 1; Figure 2 This is the fluorescence spectrum of the nitrogen-doped graphene quantum dots prepared in Example 1; Figure 3 These are morphology images and elemental mapping images of the NGQDs / Co3O4@O-SNF / ITO electrode in Example 1; Figure 4 These are comparison graphs of cyclic voltammetry curves and electrochemiluminescence signals of the SNF / ITO, Co3O4@SNF / ITO, NGQDs@SNF / ITO and NGQDs / Co3O4@SNF / ITO electrodes in PBS buffer solution containing 100 μmol / L luminol in Example 1. Figure 5 The electrochemiluminescence curves of the NGQDs / Co3O4@SNF / ITO electrode in Example 1 were obtained in a PBS buffer solution containing 100 μmol / L luminol under a nitrogen, air, or oxygen atmosphere. Figure 6 These are the cyclic voltammetry curves of the SNF / ITO, Co3O4@SNF / ITO, NGQDs@SNF / ITO and NGQDs / Co3O4@SNF / ITO electrodes in Example 1 in an air atmosphere in a PBS buffer solution containing 100 μmol / L luminol. Figure 7 These are the cyclic voltammetry curves of the SNF / ITO, Co3O4@SNF / ITO, NGQDs@SNF / ITO and NGQDs / Co3O4@SNF / ITO electrodes in Example 1 in a PBS buffer solution containing 100 μmol / L luminol under an oxygen atmosphere. Figure 8 These are the cyclic voltammetry curves of the SNF / ITO, Co3O4@SNF / ITO, NGQDs@SNF / ITO and NGQDs / Co3O4@SNF / ITO electrodes in Example 1, in a PBS buffer solution containing 100 μmol / L luminol, under a nitrogen atmosphere. Figure 9 This is an electrochemiluminescence intensity diagram of the NGQDs / Co3O4@SNF / ITO electrode in Example 1 in PBS buffer solutions containing 100 μmol / L luminol with different capture agents. Figure 10 The electrochemiluminescence curves of the NGQDs / Co3O4@SNF / ITO electrode in Example 1 were obtained in a PBS buffer solution containing 100 μmol / L luminol and 100 μmol / L hydrogen peroxide. Figure 11 The different electrodes progressively modified in Example 1 were used in 2.5 mmol / L Fe(CN)6 3- / 4- Cyclic voltammetry curves in the figure; Figure 12 The different electrodes progressively modified in Example 1 were used in 2.5 mmol / L Fe(CN)6 3- / 4- Electrochemical impedance spectroscopy in the image; Figure 13 These are the electrochemiluminescence potential curves of different electrodes progressively modified in Example 1 in a PBS buffer solution containing 100 μmol / L luminol; Figure 14 These are the electrochemiluminescence time curves of different electrodes progressively modified in Example 1 in a PBS buffer solution containing 100 μmol / L luminol; Figure 15 This is an optimized diagram of electrodeposition time for NGQDs and Co3O4 in the NGQDs / Co3O4@SNF / ITO electrode in Example 1, as well as an optimized diagram of incubation time for interleukin-1β antibody and interleukin-1β antigen. Figure 16 The images show the electrochemiluminescence curves and the linear relationship between the electrochemiluminescence intensity and the logarithm of the interleukin-1β concentration obtained after incubating the BSA / Ab / NGQDs / Co3O4@SNF / ITO electrode with different concentrations in Example 1. Figure 17 This is an electrochemiluminescence intensity diagram measured after the electrochemiluminescence immunosensor of Example 1 was incubated with interferon, target and mixture; Figure 18This is an electrochemiluminescence intensity diagram measured after incubation of five parallel working electrodes with interleukin-1β; Figure 19 This is an electrochemiluminescence intensity diagram of the working electrode in Example 1 after being stored at 4°C for different numbers of days and then incubated with interleukin-1β. Detailed Implementation
[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Modifications or equivalent substitutions made by those skilled in the art based on their understanding of the technical solutions of this invention, without departing from the spirit and scope of the invention, should be covered within the protection scope of this invention.
[0031] Unless otherwise specified, all raw materials used in the following specific embodiments were purchased commercially and used directly without special processing. The main raw materials used in the embodiments are as follows: Interleukin-1β (IL-1β): Beijing Keyue Zhongkai Biotechnology Co., Ltd.; Interleukin-1β antibody: Beijing Keyue Zhongkai Biotechnology Co., Ltd.; Potassium ferricyanide (K3[Fe(CN)6]): Shanghai Aladdin Biotechnology Co., Ltd.; Potassium ferrocyanide (K4[Fe(CN)6]): Shanghai Aladdin Biotechnology Co., Ltd.; Disodium hydrogen phosphate dodecahydrate (NaH2PO4·12H2O): Shanghai Aladdin Biotechnology Co., Ltd.; Hexadecyltrimethylammonium bromide (CTAB): Shanghai Aladdin Biotechnology Co., Ltd.; Tetraethyl silicate (TEOS): Shanghai Aladdin Biotechnology Co., Ltd.; Sodium nitrate (NaNO3): Shanghai Aladdin Biotechnology Co., Ltd.; Ferrocene Methanol (FcMeOH): Shanghai Aladdin Biotechnology Co., Ltd.; Cobalt sulfate heptahydrate (CoSO4·7H2O): Shanghai Aladdin Biotechnology Co., Ltd. (3-Epoxypropyl)trimethoxysilane (GPTMS): Shanghai Aladdin Biotechnology Co., Ltd.; Luminol: Shanghai Aladdin Biotechnology Co., Ltd. Hydrogen peroxide (H2O 2） Shanghai Aladdin Biotechnology Co., Ltd. Ammonia water (NH3·H2O): Shanghai Aladdin Biotechnology Co., Ltd.; Sodium acetate (NaAc): Shanghai Aladdin Biotechnology Co., Ltd.; 3,3',5,5'-Tetramethylbenzidine (TMB): Shanghai Aladdin Biotechnology Co., Ltd.; Glacial acetic acid (HAc): Hangzhou Gaojing Fine Chemical Co., Ltd.; 1-Aminopyrene: Shanghai Maclean Biochemical Technology Co., Ltd.; Catalase (CAT): Shanghai Maclean Biochemical Technology Co., Ltd.; 3-Aminopropyltriethoxysilane (APTES): Shanghai Maclean Biochemical Technology Co., Ltd.; Potassium hydrogen phthalate (KHP): Shanghai Maclean Biochemical Technology Co., Ltd.; Ruthenium trichloride (Ru(NH3)6Cl3): Sigma-Aldrich Trading GmbH, Germany.
[0032] In the following examples and tests, the concentration of the PBS buffer solution containing 100 μM luminol was 0.01 mol / L, and the pH value was 7.4.
[0033] Example 1: This invention provides an electrochemiluminescence immunosensor for detecting interleukin-1β, comprising a working electrode, a reference electrode, and a counter electrode. The working electrode includes a substrate electrode and a surface-modified non-phosphorylated phosphoprotein (SNF). The SNF contains a composite nanozyme comprising Co3O4 nanoparticles and NGQDs within its nanopores, and the outer surface of the SNF is modified with epoxy groups, through which anti-interleukin-1β antibodies are covalently immobilized. The detection system of the electrochemiluminescence immunosensor is a luminol / dissolved oxygen system. The reference electrode is selected from Ag / AgCl electrode (saturated KCl), saturated calomel electrode, or Hg / HgO electrode; the counter electrode is selected from platinum wire or platinum sheet; the substrate electrode is selected from indium tin oxide (ITO) electrode, glassy carbon electrode, fluorine-doped tin oxide electrode, gold electrode, screen-printed electrode, graphite electrode, or carbon fiber electrode. In this example, the reference electrode is an Ag / AgCl electrode (saturated KCl); the counter electrode is a platinum wire; and the substrate electrode is an ITO electrode.
[0034] The above-mentioned method for preparing the working electrode includes the following steps: (1) Preparation of NGQDs: A hydrothermal synthesis method was adopted. 2 mg / mL of 1-aminopyrene was added to 20 mL of 0.4 M ammonia water, mixed evenly, and then transferred to a polytetrafluoroethylene liner. The mixture was placed in a reaction vessel and hydrothermally reacted at 200 °C for 8 h. After the reaction, the reaction solution was filtered through a 0.22 μmol / L filter membrane to remove large particles, resulting in a reddish-brown solution. The solution was dialyzed for 24 h using dialysis bags with molecular weight cutoffs of 500 Da and 3500 Da, respectively, to remove unreacted small molecules and byproduct salts. The dialysate was freeze-dried to obtain NGQDs powder. The NGQDs solid powder was then dispersed in deionized water and ultrasonically treated to ensure uniform dispersion, thus obtaining a solution containing NGQDs.
[0035] (2) Preparation of SNF / ITO electrode: 160 mg of CTAB was added to a mixed solution of 30 mL of ethanol and 70 mL of water and stirred for 5 min until completely dissolved. Under slow stirring, 100 μL of 10% ammonia solution and 80 μL of tetraethyl orthosilicate (TEOS) were added sequentially until the solution became transparent and bubble-free. Then, the pretreated ITO electrode was placed in the solution and reacted in a water bath at 60 °C for 24 h. After the reaction was completed, the electrode was rinsed with a large amount of ultrapure water and then aged at 100 °C for 12 h to obtain the SNF modified electrode, denoted as SM@SNF / ITO.
[0036] (3) Preparation of O-SNF / ITO electrode: The SM@SNF / ITO electrode was immersed in a 2.26 mmol / L solution of 3-glycidyl etheroxypropyltrimethoxysilane (GPTMS) in ethanol and reacted at room temperature in the dark for 1 h. After washing with deionized water and drying with nitrogen, an epoxy-functionalized electrode was obtained. The above electrode was then immersed in a 0.1 M hydrochloric acid-ethanol solution and stirred for 5 min to remove residual micelles in the pores, resulting in an O-SNF / ITO electrode with open pores.
[0037] (4) Preparation of Co3O4@O-SNF / ITO electrode: The O-SNF / ITO electrode was placed in a 0.2 mol / L CoSO4 solution, and a voltage of +1.5 V was applied for 10 s. After removal, it was rinsed with a large amount of flowing ultrapure water and dried with nitrogen to obtain the Co3O4@O-SNF / ITO electrode.
[0038] (5) Preparation of NGQDs / Co3O4@O-SNF / ITO electrode: The Co3O4@O-SNF / ITO electrode was first subjected to a constant voltage of -0.5 V for 10 s, and then immersed in an NGQDs solution with a concentration of 0.5 mg / mL. A voltage of +0.8 V was applied for 180 s. After removal, it was rinsed with ultrapure water and dried with nitrogen to obtain the NGQDs / Co3O4@O-SN / ITO electrode with Co3O4 and NGQDs confined in the pores.
[0039] (6) Preparation of BSA / Ab / NGQDs / Co3O4@O-SNF / ITO electrode: 20 μmol / L interleukin-1β antibody solution was dropped onto the surface of the NGQDs / Co3O4@O-SNF / ITO electrode and incubated at 4 °C for 90 min. Unbound antibody was removed by thorough washing with 0.01 M, pH 7.4 phosphate buffer and dried with nitrogen to obtain the Ab / NGQDs / Co3O4@O-SNF / ITO electrode. Subsequently, 0.5% BSA solution was dropped onto the electrode surface and reacted at room temperature for 10 min to block non-specific adsorption sites, thus obtaining the final working electrode: BSA / Ab / NGQDs / Co3O4@O-SNF / ITO electrode.
[0040] In step (2), the pretreatment process of the ITO electrode is as follows: the ITO electrode is soaked in 1 M NaOH overnight, then sonicated with acetone and ethanol for 30 min each, and finally sonicated with deionized water three times for 10 min each time.
[0041] The procedure for detecting interleukin-1β in serum using the above-mentioned BSA / Ab / NGQDs / Co3O4@O-SNF / ITO electrode via electrochemiluminescence is as follows: Serum samples were diluted 50-fold with 0.01 mol / L, pH 7.0 phosphate buffer, and interleukin-1β standard was added to prepare test sample solutions with concentrations of 100 fg / mL, 1 pg / mL, 10 pg / mL, 0.1 ng / mL, 1 ng / mL, 10 ng / mL, and 100 ng / mL. The test sample solutions were then dropped onto the surface of the immunosensing working electrode and incubated at 4 ℃ for 90 min. The solution was then used with 0.01 M, pH 7.4... Unbound antigens were removed by rinsing with phosphate buffer to obtain the test electrode. The test electrode, along with the reference electrode and the counter electrode, was placed in a luminol / dissolved oxygen detection solution. A scanning voltage was applied, and the electrochemiluminescence signal was recorded. The degree of reduction in the electrochemiluminescence signal was detected to quantify interleukin-1β. The scanning voltage range was -1.0 V to 0.8 V.
[0042] The fluorescence properties and UV-Vis absorption spectra of the NGQDs prepared in Example 1 were characterized, and the results are as follows: Figure 1 and Figure 2 As shown, Figure 1 The illustration shows a photograph of NGQDs under 365 nm ultraviolet light. These results demonstrate the successful synthesis of NGQDs with excellent fluorescence properties.
[0043] The morphology of the NGQDs / Co3O4@O-SNF / ITO electrode prepared in Example 1 was characterized, and the results are as follows: Figure 3 As shown. Figure 3 As can be seen, the electrode consists of an SNF layer, an ITO layer, and a glass layer from top to bottom. The surface of the SNF layer is intact and free of cracks, with worm-like pores evenly distributed. No large particles are observed to aggregate and obstruct the pores on the surface, indicating that the nanozymes are all confined inside the pores. This proves that the present invention has successfully prepared the target working electrode, and that Co3O4 and NGQDs are effectively confined inside the pores of the SNF.
[0044] To verify the synergistic effect of Co3O4 / NGQDs composite nanozymes, the SNF / ITO ECL signals of confined single nanozymes NGQDs, Co3O4, and Co3O4 / NGQDs composite nanozymes in the luminol / dissolved oxygen system were compared. The results are as follows: Figure 4 As shown, the unconfined SNF / ITO electrode exhibits extremely low oxygen reduction peak current at -1 V and luminol oxidation peak current at +0.8 V, with an ECL signal of only 842 au. After confining single NGQDs and Co3O4, the ECL signals are improved, producing signal intensities of 10751 au and 2289 au, respectively. The corresponding oxygen reduction peak current and luminol oxidation peak current in the CV plot are significantly improved, and the onset potential of oxygen reduction under the catalysis of NGQDs is also significantly advanced. After confining the composite nanozyme, the ECL signal intensity (26770 au) generated by the NGQDs / Co3O4@SNF / ITO electrode is significantly higher than the sum of the signals of the Co3O4@SNF / ITO (2289 au) and NGQDs@SNF / ITO (10751 au) electrodes, proving that the Co3O4 / NGQDs composite nanozyme produces a significant synergistic catalytic enhancement effect, rather than a simple additive effect.
[0045] The mechanism by which Co3O4 / NGQDs composite nanozymes enhance the electrochemiluminescence of the luminol / dissolved oxygen system was investigated. Figure 5As shown, ECL tests were conducted in N2, air, and O2 saturated atmospheres, respectively. When the system contained no DO, there was almost no ECL signal, but as the DO content in the system increased, the ECL signal was significantly enhanced. Figures 6 to 8 As shown, the oxygen reduction peak current at -0.5 V gradually increases with increasing DO content, proving that DO is a co-reactant in this system. Without O2 generating reactive oxygen species to react with luminol radicals, the system cannot produce luminescence. The types of reactive oxygen species generated during this reaction are verified using free radical scavengers, such as... Figure 9 As shown, the measured ECL intensity did not decrease significantly after the addition of the ∙OH scavenger TBA. However, the ECL intensity decreased by 68% after the addition of CAT. Furthermore, the signal was almost completely quenched after the addition of the superoxide anion radical scavenger BQ, indicating that the main reactive oxygen species generated in the system were superoxide anion radicals. Simultaneously, during oxygen reduction, O2 underwent a two-electron transfer reaction to generate H2O2. H2O2 then regenerated superoxide anion radicals during anodic oxidation, which reacted with the oxidized luminol. To verify the significant catalytic effect of Co3O4 on the generated H2O2, ECL tests were performed on the confined single nanozyme and composite nanozyme SNF / ITO electrodes in the luminol / hydrogen peroxide system, as shown below. Figure 10 As shown, the electrode with Co3O4 confined alone has a signal 50% higher than the electrode with NGQDs confined alone, and the combined effect of the two has an even higher signal, proving that Co3O4 mainly plays a role in catalyzing the production of reactive oxygen species from H2O2. Figure 5 and Figure 10 As can be seen, the composite nanozyme system of this invention can generate a high-intensity ECL signal in the presence of dissolved oxygen, and its intensity is even better than that of the single-component nanozyme in the comparative experiment with the addition of exogenous H2O2. This highlights the unique advantage of this invention in constructing a green and efficient detection system using endogenous dissolved oxygen.
[0046] To verify the success of the stepwise modification process of the electrochemiluminescence immunosensor of the present invention, different electrodes that were stepwise modified in Example 1 were subjected to 2.5 mmol / L Fe(CN)6. 3- / 4- The electrochemical performance in the middle, the results are as follows Figure 11 and Figure 12 As shown. Figure 11 In the process, as epoxy groups, antibodies, BSA and interleukin-1β were modified on the surface of NGQDs / Co3O4@SNF / ITO electrodes, the steric hindrance of the electrode surface gradually increased, making diffusion mass transfer and electron transfer on the electrode surface increasingly difficult, resulting in a gradual decrease in current signal, which verified the successful modification of the sensor at each step. Figure 12The gradually increasing Rct value further verifies the successful modification of the sensor at each step.
[0047] To verify the feasibility of using the electrochemiluminescence immunosensor of the present invention for interleukin-1β detection, electrochemiluminescence tests were performed on different electrodes progressively modified in Example 1, and the results are as follows: Figure 13 and Figure 14 As shown in the figure, the ECL signal is significantly enhanced after the addition of NGQDs and Co3O4 to the electrically limiting domain. This is attributed to the formation of a composite nanozyme, which efficiently catalyzes the reaction of luminol with dissolved oxygen, generating more luminol radicals and reactive oxygen species, thereby greatly enhancing the electrochemiluminescence signal. When epoxy groups and antibodies are modified sequentially, the ECL signal of the Ab / NGQDs / Co3O4@O-SNF / ITO electrode decreases. This is because antibody immobilization creates a steric hindrance effect, hindering luminol and oxygen from entering the nanochannels and inhibiting electron transfer on the electrode surface, leading to a decrease in the ECL signal. After blocking non-specific sites with BSA, the non-conductive protein further hinders the diffusion of the luminescent probe and electron transfer, causing the ECL signal to continue to decrease. When interleukin-1β is introduced, the antigen and antibody specifically bind, further increasing the steric hindrance on the electrode surface, hindering probe transport and electron transfer, ultimately causing a further decrease in the ECL signal. These results confirm that the immunosensor has been successfully constructed and can achieve specific detection of interleukin-1β.
[0048] To obtain optimal detection conditions, the deposition time of Co3O4, the electrophoresis time of NGQDs, the incubation time of the antibody, and the incubation time of the antigen were tested. Different working electrodes were obtained by changing the deposition times of Co3O4 and NGQDs in Example 1, and detection was performed. The results are as follows: Figure 15 As shown in (A) and (B), the ECL signal exhibits a trend of first increasing and then decreasing. The optimal deposition time for Co3O4 is 10 s, and the optimal electrophoresis time for NGQDs is 180 s. Changing the incubation time of the antibody and antigen in Example 1 yielded the following results: Figure 15 As shown in (C) and (D), it can be seen that as time increases, both show a trend of first decreasing and then stabilizing, indicating that the modification amount of antibody and antigen has reached the maximum, the optimal incubation time is 30 min, and the antigen incubation time is 90 min.
[0049] The electrochemiluminescence immunosensor constructed in Example 1 was used to detect different concentrations of IL-1β, and the results are as follows: Figure 16As shown, the ECL signal gradually decreased with increasing IL-1β concentration. Within the range of 100 fg / mL to 100 ng / mL, the ECL intensity showed a good linear relationship with the logarithm of IL-1β concentration. The LOD value was calculated to be 28 fg / mL based on the corresponding linear regression equation.
[0050] The anti-interference performance of the electrochemiluminescence immunosensor constructed in Example 1 was tested. Common ions, electroactive substances, and common inflammatory markers in serum were selected as interfering agents. The results are as follows: Figure 17 As shown, when IL-1β is absent in the test solution, there is almost no decrease in the ECL signal. The decrease in the ECL signal only occurs in solutions containing IL-1β or a mixture of IL-1β and interfering substances, indicating that the sensor prepared in this invention has excellent selectivity and anti-interference capabilities.
[0051] To examine the reproducibility of the electrochemiluminescence immunosensor constructed in Example 1, five identical working electrodes were prepared in parallel and used for the detection of 10 ng / mL interleukin-1β. The results are shown in Figure 18. The obtained electrochemiluminescence signals have good consistency, with a relative standard deviation (RSD) of 2.3%, indicating that the sensor prepared in this invention has good reproducibility.
[0052] To examine the long-term stability of the electrochemiluminescence immunosensor constructed in Example 1, the working electrode from Example 1 was refrigerated at 4°C, and one electrode was taken out daily to detect 10 ng / mL interleukin-1β. The results are as follows: Figure 19 As shown, the detection signal decreased by only 7.5% from day 1 to day 5, indicating that the sensor has good long-term stability.
[0053] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.
[0054] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.
Claims
1. An electrochemiluminescent immunosensor for detecting interleukin-1β, characterized in that, Includes the working electrode, reference electrode, and counter electrode; The working electrode includes a base electrode and an SNF modified on the surface of the base electrode. The SNF has nanopores loaded with a composite nanozyme containing Co3O4 nanoparticles and NGQDs, and the outer surface of the SNF is modified with epoxy groups, and an anti-interleukin-1β antibody is covalently fixed through the epoxy groups. The detection system of the electrochemiluminescence immunosensor is a luminol / dissolved oxygen system.
2. The electrochemiluminescent immunosensor for detecting interleukin-1β according to claim 1, characterized in that, The distance between the Co3O4 nanoparticles and the adjacent NGQDs in the nanopores is no greater than 3 nm.
3. An electrochemiluminescent immunosensor for detecting interleukin-1β according to any one of claims 1 to 2, characterized in that, The pH value of the detection system is 6.8~7.
6.
4. A method for preparing an electrochemiluminescent immunosensor for detecting interleukin-1β, characterized in that, Includes the following steps: (1) Modify SNF on the surface of the substrate electrode to obtain an SNF-modified electrode, immerse it in a silane solution containing epoxy groups to react, and obtain an O-SNF-modified electrode with epoxy groups on the outer surface. (2) The O-SNF modified electrode was immersed in a solution containing CoSO4 for electrodeposition to obtain Co3O4@O-SNF modified electrode; (3) The Co3O4@O-SNF modified electrode was immersed in a solution containing NGQDs for electrophoresis treatment to obtain the NGQDs / Co3O4@O-SNF modified electrode; (4) The NGQDs / Co3O4@O-SNF modified electrode was incubated with a buffer solution containing anti-interleukin-1β antibody, and the non-specific sites were blocked with bovine serum albumin solution to obtain the working electrode.
5. The method for preparing an electrochemiluminescent immunosensor for detecting interleukin-1β according to claim 4, characterized in that, In step (2), the concentration of CoSO4 in the CoSO4-containing solution is 0.02~0.2 mol / L; the electrodeposition voltage is +1.0 ~ +2.0 V, and the deposition time is 1 ~ 20 s.
6. The method for preparing an electrochemiluminescent immunosensor for detecting interleukin-1β according to claim 4, characterized in that, In step (3), the electrophoresis treatment potential is +0.4 ~ +1.0 V, and the electrophoresis time is 30 ~ 180 s.
7. The method for preparing an electrochemiluminescent immunosensor for detecting interleukin-1β according to claim 4, characterized in that, In step (1), the concentration of the silane containing the epoxy group in the silane solution is 2.0 ~ 3.0 mmol / L.
8. The method for preparing an electrochemiluminescent immunosensor for detecting interleukin-1β according to claim 4, characterized in that, In step (4), the incubation temperature is 0~5℃ and the time is 0.5~3 h.
9. The application of an electrochemiluminescence immunosensor in the detection of interleukin-1β for non-diagnostic purposes, characterized in that, The electrochemiluminescence immunosensor is the electrochemiluminescence immunosensor as described in any one of claims 1 to 3, comprising the following steps: incubating the working electrode of the electrochemiluminescence immunosensor with a test solution containing interleukin-1β; placing the incubated working electrode together with the reference electrode and the counter electrode in a luminol / dissolved oxygen system detection solution; using dissolved oxygen as a co-reactant; applying a scanning voltage; and quantitatively analyzing interleukin-1β by detecting the degree of reduction in the electrochemiluminescence signal.
10. The application of the electrochemiluminescence immunosensor according to claim 9 in the detection of interleukin-1β for non-diagnostic purposes, characterized in that, The luminol concentration in the luminol / dissolved oxygen system detection solution is 10~200 μmol / L.