Electrochemiluminescence immunosensor for detecting interleukin-1 beta and preparation method and application thereof

Abstract

The application discloses an electrochemiluminescence immunosensor for detecting interleukin-1 beta and a preparation method and application thereof, and comprises a three-electrode system of a working electrode, a reference electrode and a counter electrode; the working electrode is a modified electrode of CoAl layered double hydroxide, is loaded with platinum nanoparticles after being etched by alkali, and is obtained by fixing interleukin-1 beta antibodies and blocking non-specific sites; and a solution containing luminol is used as an electrolyte during detection of the immunosensor. The working electrode of the immunosensor takes oxygen-rich vacancy CoAl layered double hydroxide as a substrate, combines with platinum nanoparticles to realize EMSI effect, significantly enhances the electrochemiluminescence signal of a luminol-dissolved oxygen system, realizes quantitative detection of IL-1 beta by combining with specific antigen-antibody recognition, and has the characteristics of strong anti-interference ability, fast detection speed and good stability.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, specifically to an electrochemiluminescent immunosensor for detecting interleukin-1β, its preparation method, and its application. Background Technology

[0002] Interleukin-1β (IL-1β), an important pro-inflammatory cytokine, is primarily derived from immune cells such as monocytes and macrophages. It plays a crucial role in many physiological and pathological processes, including initiating immune responses, regulating inflammatory reactions, and promoting tissue repair after injury. For instance, the detection of the pro-inflammatory cytokine interleukin-1β (IL-1β) has significant clinical value in obstetrics, as abnormally elevated levels can reflect the pathological progression of various pregnancy complications.

[0003] In premature rupture of membranes (PROM), serum IL-1β > 0.38 ng / mL showed a sensitivity of 76.5% and a specificity of 72.6% for diagnosing chorioamnionitis (CAM). Furthermore, elevated umbilical cord blood IL-1β levels in newborns of mothers with intrauterine infections were positively correlated with decreased Apgar scores, neurological damage, and renal dysfunction. For preterm birth prediction, umbilical cord blood IL-1β levels significantly increased in maternal inflammatory responses ≥ stage 2. Combined with IL-6 and IL-8, it could predict 92.3% of fetal inflammatory responses and was associated with long-term complications such as intraventricular hemorrhage in preterm infants. In patients with fetal growth restriction (FGR), elevated placental IL-1β expression was negatively correlated with neonatal weight and length, and it induced trophoblast apoptosis by regulating the Bcl-2 / Caspase-3 pathway.

[0004] Furthermore, serum IL-1β levels in patients with gestational diabetes mellitus (GDM) are associated with glycemic control (higher levels are seen in patients with HbA1c ≥7%) and insulin resistance; dynamic monitoring can assess the efficacy of anti-inflammatory treatment. In pregnancy with systemic lupus erythematosus (SLE), persistently high IL-1β expression indicates a risk of disease activity, necessitating enhanced immune regulation. In summary, IL-1β testing, by quantifying the intensity of inflammation, provides crucial evidence for the early identification, risk stratification, and timing of intervention for the aforementioned complications.

[0005] Among the methods for detecting IL-1β, enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay, and quantitative real-time polymerase chain reaction (qPCR) are relatively common. While ELISA is widely used due to its relatively simple operation and low cost, it has significant drawbacks: it takes a long time, generally 4-8 hours; its sensitivity is not ideal, with detection limits mostly at the pg / mL level; and it cannot provide real-time detection. Although fluorescence immunoassay has improved sensitivity, it is easily affected by background fluorescence, and the stability of fluorescent probes is poor, affecting the accuracy of the results. qPCR mainly targets IL-1β gene expression and cannot directly reflect its protein level. Furthermore, its complex operation and high instrument cost limit its application in rapid clinical testing. These problems make it difficult for existing methods to meet the clinical need for accurate and rapid detection of low concentrations of IL-1β.

[0006] Electrochemiluminescence (ECL) immunoassay technology holds immense potential in the field of biomarker detection, boasting advantages such as ultra-high sensitivity, good selectivity, and a wide linear range. Luminol is a commonly used ECL luminescent reagent, and the catalytic effect of nanomaterials can significantly enhance its luminescence efficiency. Recent studies have discovered that electron-metal-support interaction (EMSI) can effectively modulate the surface electronic state and catalytic active centers of metal nanomaterials, thereby greatly enhancing their catalytic performance. This provides a new approach to improving the sensitivity of ECL detection.

[0007] Layered bimetallic hydroxides (LDHs) are nanomaterials with a unique two-dimensional layered structure, and their chemical composition and interlayer ions are highly tunable. Platinum nanoparticles (PtNPs) possess excellent electrocatalytic activity and chemical stability, and are widely used in electrochemical catalytic reactions, effectively promoting the luminol-dissolved oxygen ECL reaction; however, there are few reports on their application in current technologies.

[0008] CN117129543A discloses a sensor based on layered double metal hydroxides, its preparation method, and its application. The preparation method includes the following steps: S1, gold nanoparticle solution, ferrocene@magnesium-aluminum layered double hydroxide solution, and gold nanoparticle solution are sequentially added to a glassy carbon electrode and dried to obtain sample A; S2, a 0.5μM-1.2μM thiolized SHcDNA probe solution is added to sample A and incubated to obtain sample B; the thiolized SHcDNA probe solution is obtained by diluting with PBS buffer at pH 6.5-8; S3, sample B is incubated with mercaptoethanol solution to obtain sample C; S4, sample C is incubated with bladder cancer marker solution for 25-50 minutes to obtain sample D; S5, a thiolized MBcDNA probe@gold nanoparticle solution is added to sample D and incubated for 80-90 minutes to obtain the sensor. This sensor can be used for sensitive, accurate, and specific detection of bladder cancer markers.

[0009] To address the problems of insufficient sensitivity, cumbersome operation, and weak anti-interference ability of IL-1β detection methods, this paper provides a new method and approach for rapid and accurate detection, which is of great significance to both theoretical research and practical application. Summary of the Invention

[0010] This invention addresses the problems of insufficient sensitivity, cumbersome operation, and weak anti-interference ability of existing IL-1β detection methods by providing an electrochemiluminescence immunosensor for detecting interleukin-1β. By designing a composite structure of oxygen-vacancy-rich CoAl layered double metal hydroxide (LDH-Ov) and platinum nanoparticles (PtNPs), the electrochemiluminescence signal of the luminol-dissolved oxygen system is significantly amplified by the electronic metal-carrier interaction between the two. Combined with antigen-antibody specific recognition, ultrasensitive and highly selective detection of IL-1β is achieved.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0012] An electrochemiluminescent immunosensor for detecting interleukin-1β includes a three-electrode system comprising a working electrode, a reference electrode, and a counter electrode.

[0013] The working electrode is a modified electrode of CoAl layered double metal hydroxide, which is loaded with platinum nanoparticles after alkaline etching, and then immobilized with interleukin-1β antibody and blocked with non-specific sites.

[0014] This invention uses oxygen-vacancy-rich double hydroxide (LDH-Ov) as a support, deposits PtNPs on its surface, and utilizes EMSI to enhance the catalytic efficiency of the catalyst. It then leverages the sensitized electrochemiluminescence signal of the luminol-dissolved oxygen system, combined with an immunorecognition interface, to achieve highly sensitive detection of IL-1β. Based on this, an immunosensor is constructed using LDH-Ov loaded with PtNPs, enhanced ECL signal via EMSI, and an immunosensor to detect IL-1β. This provides a new method and approach for rapid and accurate detection of IL-1β, achieving ultra-high sensitivity detection of the inflammatory marker interleukin-1β (IL-1β). This technology has significant theoretical research value and practical application prospects, and can be widely used in the early diagnosis and monitoring of inflammatory diseases such as rheumatoid arthritis and gout.

[0015] The immunosensor uses a solution containing luminol as the electrolyte during detection.

[0016] The working electrode preparation process includes:

[0017] Step 1: Modify the electrode surface with CoAl layered bimetallic hydroxide (CoAl-LDH) to obtain a CoAl-LDH modified electrode, and then obtain an LDH-Ov modified electrode rich in oxygen vacancies by alkaline etching.

[0018] Step 2: Immerse the LDH-Ov modified electrode in a solution containing a platinum precursor and electrodeposit to obtain a Pt@LDH-Ov modified electrode with platinum nanoparticles loaded on its surface.

[0019] Step 3: Drop a buffer solution containing IL-1β antibody onto the surface of the Pt@LDH-Ov modified electrode and incubate it. After incubation with bovine serum albumin (BSA) aqueous solution to block non-specific sites, the working electrode is obtained.

[0020] LDH-Ov, obtained through alkaline etching, contains a large number of oxygen vacancies. When these vacancies combine with in-situ electrodeposited PtNPs, a strong EMSI effect is formed. This effect drives the electrocatalytic reduction of dissolved oxygen by PtNPs, generating reactive oxygen species (ROS), which in turn significantly enhances the ECL signal of luminol. Furthermore, the layered structure of LDH-Ov can serve as a stable substrate for immobilizing PtNPs. Antibodies can then be bound via Pt-NH2 bonds. Utilizing the specific recognition ability of antigens and antibodies, the change in electrode ECL signal caused by the binding of the recognition antibody to IL-1β ultimately enables the quantitative detection of IL-1β.

[0021] Step 1, modifying the electrode surface with CoAl layered bimetallic hydroxide, specifically includes:

[0022] The electrode was immersed in a precursor solution containing cobalt nitrate, aluminum nitrate, urea and ammonium fluoride, and reacted in a water bath at 60-100℃ for 6-8 h. After washing and drying, the CoAl-LDH modified electrode was obtained.

[0023] The precursor solution contains cobalt nitrate at a molar concentration of 0.1-0.8 mM, aluminum nitrate at a molar concentration of 0.05-0.5 mM, urea at a molar concentration of 1-6 mM, and ammonium fluoride at a molar concentration of 0.5-3 mM.

[0024] The 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.

[0025] The alkaline etching process includes: immersing the CoAl-LDH modified electrode in NaOH solution, removing it, washing it with deionized water, and drying it with nitrogen to obtain the LDH-Ov modified electrode;

[0026] The NaOH solution concentration is 1-10 mol / L, and the soaking is carried out at room temperature for 15-45 min. The solvent used for NaOH includes any one or more of ethanol, water, and diethyl ether.

[0027] The solution containing the platinum precursor is an aqueous solution of chloroplatinic acid with a concentration of 0.3-0.7 mM;

[0028] In step 2, the electrodeposition voltage is constant from -1.0 to 0 V, the electrodeposition time is 1 to 10 seconds, and the temperature is room temperature.

[0029] The molar concentration of the buffer solution containing IL-1β antibody is 1~20 μg / mL; the solvent used includes any one or more of sodium chloride-ethylenediaminetetraacetic acid-tris(hydroxymethyl)aminomethane buffer (STE) and tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid buffer (TE); the incubation conditions are 0-5℃ for 0.5-2 h.

[0030] The aqueous solution of bovine serum albumin has a concentration of 0.2-2% (w / w) and is incubated at 0-5°C for 5-15 min.

[0031] The molar concentration of luminol in the detection solution is 10 ~ 300 μmol / L; within this concentration range, the ECL signal is kept within a suitable absolute value range, which makes this detection technology more sensitive and economical.

[0032] The pH of the test solution is 6-8. This pH of the test solution can avoid inhibiting the deprotonation of luminol, resulting in high electrochemiluminescence intensity and sensitivity, and high detection stability. Preferably, the pH of the test solution is 6.5-8, and more preferably, it is 6.5-7.5.

[0033] This invention also provides a method for preparing the electrochemiluminescent immunosensor for detecting interleukin-1β, comprising the following steps:

[0034] Step 1: Modify the electrode surface with CoAl layered bimetallic hydroxide to obtain CoAl-LDH modified electrode, and then obtain LDH-Ov modified electrode rich in oxygen vacancies by alkaline etching.

[0035] Step 2: Immerse the LDH-Ov modified electrode in a solution containing a platinum precursor and electrodeposit to obtain a Pt@LDH-Ov modified electrode with platinum nanoparticles loaded on its surface.

[0036] Step 3: Drop a buffer solution containing IL-1β antibody onto the surface of the Pt@LDH-Ov modified electrode and incubate it. After incubation with bovine serum albumin aqueous solution to block non-specific sites, the working electrode is obtained.

[0037] Step 5: The immunosensor is obtained by forming a three-electrode system with the working electrode, the counter electrode and the reference electrode. The immunosensor uses a solution containing luminol as the electrolyte during detection.

[0038] This invention also provides a method for electrochemiluminescence detection of interleukin-1β, wherein the immunoelectrode is incubated with a test solution and then rinsed to obtain the test electrode; the test electrode is used as the working electrode, forming a three-electrode system with a reference electrode and a counter electrode, and an electrolyte solution containing luminol is used as the detection solution for electrochemiluminescence signal detection. These detection methods are not intended for diagnostic or therapeutic purposes, such as related mechanism research.

[0039] The detection principle is as follows:

[0040] (1) EMSI Enhances ECL Signal: After alkaline etching, LDH generates a large number of oxygen vacancies. These oxygen vacancies act as electron donors and form strong electron-electron interactions (EMSI) with PtNPs, enriching the PtNPs surface with electrons (Pt... δ- This significantly enhances its ability to catalyze the reduction of dissolved oxygen, generating a large number of superoxide anion radicals (O2). ·- O2 ·- With luminol anion radical (L ·- The reaction produces excited-state 3-aminophthalate (AP) ions. 2-* When it returns to the ground state, it releases a strong ECL signal.

[0041] (2) Antigen-antibody specific recognition: When IL-1β is present in the test solution, IL-1β specifically binds to the antibody on the surface of the working electrode to form an immune complex. This complex has a large steric hindrance, which hinders the diffusion of luminol molecules and dissolved oxygen to the electrode surface, resulting in the ECL signal decreasing with the increase of IL-1β concentration. The quantitative detection of IL-1β can be achieved by measuring the signal reduction value.

[0042] The concentration of interleukin-1β in the test solution is below 10 μg / mL; preferably, the concentration of interleukin-1β is below 100 ng / mL. The linear range for IL-1β detection by this method is 1 fg / mL to 100 ng / mL, and the limit of detection is 0.47 fg / mL; the intra-assay relative standard deviation (RSD) of five consecutive parallel experiments is ≤1.3%. In actual testing, due to the high concentration of IL-1β specific antigen or the presence of many interfering substances in the serum sample, the sample can be diluted with an electrolyte solution, such as by 1-100 times, to reduce the concentration of IL-1β specific antigen.

[0043] The incubation is carried out at 0-5℃ for 30-120 minutes.

[0044] The test solution includes one or more interfering compounds selected from glucose, inorganic salts, cytokines, and tumor markers; the total molar concentration of the interfering compounds is below 0.5 M. Because the immunosensor in this invention involves specific antigen-antibody binding, it exhibits high selectivity, is less susceptible to interference from other substances, has high detection stability, and a wide range of applications. The sensor in this invention shows a linear relationship between the logarithm of the target analyte antigen detection concentration and the target analyte antigen concentration in the range of 1 fg / mL to 100 ng / mL, with a detection limit of 0.47 fg / mL. This detection method demonstrates a wide linear range, high selectivity, high sensitivity, and low detection limit. Furthermore, the detection does not require complex pretreatment and separation processes, reducing detection costs, simplifying operation, and making it suitable for large-scale applications.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] (1) The electrochemiluminescence immunosensor in this invention utilizes the strong electronic interaction between CoAl layered double metal hydroxide rich in oxygen vacancies and platinum nanoparticles to significantly enhance the ECL signal of the luminol-dissolved oxygen system compared to the action of PtNPs alone, thereby achieving the detection of IL-1β with ultra-high sensitivity and ultra-wide linear region, which can meet the needs of trace detection of IL-1β in the early stage of inflammation.

[0047] (2) This invention utilizes alkaline etching technology to significantly increase active sites. Through alkaline etching, the originally relatively regular surface structure of LDH generates abundant defect sites and pores, greatly exposing active sites that can be anchored to platinum nanoparticles and adsorbed by reactants. Compared with untreated LDH, the alkaline-etched LDH binds more tightly and efficiently to platinum nanoparticles, allowing each active site to fully participate in the electrochemiluminescence reaction process, significantly improving the reaction rate and conversion efficiency.

[0048] (3) The reagents used in this patent are inexpensive, resulting in low detection costs. The ITO substrate electrode is inexpensive, and the working electrode is prepared by a combination of solvothermal method and in-situ electrodeposition, which does not require complex instruments and equipment. The entire detection process (including sample pretreatment, incubation and signal detection) can be completed in a short time, making it suitable for clinical point-of-care testing. Attached Figure Description

[0049] Figure 1 The images show the microstructure and characterization of the LDH / ITO or LDH-Ov / ITO electrodes prepared in Example 1. Figure 1 Image A in the image is a SEM image of the LDH / ITO electrode (the inset is a magnified view of a portion of the image). Figure 1 B in the image is a SEM image of LDH-Ov / ITO. Figure 1 C in the image represents the SEM elemental energy spectrum of LDH / ITO. Figure 1 D in the diagram represents the elemental mapping of C, Co, Al, and O in LDH / ITO.

[0050] Figure 2 These are XPS characterization data for different electrodes in Example 1. Figure 2 In the image, A represents XPS images of LDH / ITO, LDH-Ov / ITO, Pt@LDH / ITO, and Pt@LDH-Ov / ITO. Figure 2 In the image, B represents the high-resolution XPS deconvolutioned Co2p images in Pt@LDH / ITO and Pt@LDH-Ov / ITO. Figure 2 C represents the high-resolution XPS deconvolutioned images of Pt@LDH / ITO and Pt@LDH-Ov / ITO at O ​​1s.

[0051] Figure 3 The electrochemical performance of different electrodes in Example 1 is shown. Figure 3 In Figure A, the ECL response of different electrodes in the presence of luminol (100 μM) is shown. Figure 3 In the diagram, B represents the signal stability of different electrodes. Figure 3 C represents the stability and repeatability of the Pt@LDH-Ov / ITO electrode. Figure 3Figure D shows the ECL potential curves of each electrode obtained in PBS (0.01 M, pH = 7.4) containing 100 μM luminol. Figure 3 E represents the CV curves of Pt@LDH-Ov / ITO in PBS (0.01 M, pH 7.4) and luminol (100 μM) under different atmospheres (the inset shows the ECL signal of Pt@LDH-Ov / ITO under different atmospheres). Figure 3 F represents the ECL signal of Pt@LDH-Ov / ITO in different electrolytes. Figure 3 G represents the ECL signal amplification strategy of Pt@LDH-Ov / ITO.

[0052] Figure 4 Different electrodes from Example 1 were used in a solution containing 2.5 mM (1:1) [Fe(CN)6]. 3- / 4- CV curves and linear relationships obtained at different scan rates in 0.1 M KCl. Figure 4 In the middle, A represents the CV curve of the LDH-Ov / ITO electrode. Figure 4 B represents the CV curve of the Pt@LDH / ITO electrode. Figure 4 C represents the CV curve of the Pt@LDH-Ov / ITO electrode. Figure 4 D in the figure represents the linear relationship between the LDH-Ov / ITO electrodes. Figure 4 E in the figure represents the linear relationship between the Pt@LDH / ITO electrode. Figure 4 F in the figure represents the linear relationship of the Pt@LDH-Ov / ITO electrode.

[0053] Figure 5 This is the optimized ECL response of Pt@LDH-Ov / ITO in the presence of 100 μM luminol, considering different LDH etching times, PtNP deposition times, IL-1β antibody incubation times, and IL-1β antigen incubation times. Figure 5 In the figure, A represents different LDH etching times. Figure 5 B represents the optimization of deposition time for different PtNPs. Figure 5 C represents the optimized incubation time for antibodies against different IL-1β levels. Figure 5 D represents the optimization of incubation time for different IL-1β antigens.

[0054] Figure 6 The ECL signal, CV curve, and EIS curve of the immunosensors prepared with different electrodes in Example 1 are shown. Figure 6 In Figure A, ECL signals were obtained from different electrodes in PBS (0.01 M, pH = 7.4) containing 100 μM luminol. The concentration of IL-1β antibody was 10 μg / mL and the concentration of IL-1β antigen was 1 ng / mL. Figure 6 B represents the CV curves of immunosensors fabricated with different electrodes. Figure 6 C represents the EIS curves of immunosensors fabricated with different electrodes. The test solution contained 2.5 mM Fe(CN)6. 3- / 4- The 0.1 MKCl solution was scanned at a rate of 100 mV / s.

[0055] Figure 7 The calibration curves for the ECL signal, ECL intensity, and IL-1β logarithmic concentration of the Anti-IL-1β / Pt@LDH-Ov-ITO immunosensor in Example 1 are shown. Figure 7 In Figure A, the ECL signal of the immunosensor is shown at different concentrations of IL-1β. Figure 7 Figure B shows the calibration curve of ECL intensity and IL-1β logarithmic concentration of the immunosensor. The electrolyte solution is PBS (0.01 M, pH = 7.4) containing luminol (100 μM).

[0056] Figure 8 To demonstrate the anti-interference performance and stability of the immune sensor in Example 1, Figure 8 In section A, the specificity is for different antigens, with CRP and PIGF concentrations at 10 ng / mL, and Glu and K... + NO3 - AA, Na + The concentration is 1 mM, Cl - The concentrations were 1 mM for IL-1β, 1 mM for IL-6, TNF-α, IFN-γ, and NGAL, and 1 ng / mL for all of them. Figure 8 In the middle B, the signal stability of the immunosensor is given, with an IL-1β concentration of 100 pg / mL. Figure 8 In the figure, C represents the repeatability of the immunosensor, the IL-1β concentration is 1.0 ng / mL, the detection solution is PBS (0.01 M, pH = 7.4) containing 100 μM luminol, PMT = 750 V, and the error bars represent the standard deviation of three measurements performed under the same experimental conditions. Detailed Implementation

[0057] 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.

[0058] 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:

[0059] Potassium ferricyanide: Shanghai Aladdin Biotechnology Co., Ltd.;

[0060] Potassium ferrocyanide: Shanghai Aladdin Biotechnology Co., Ltd.;

[0061] Bovine serum albumin: Shanghai Aladdin Biotechnology Co., Ltd.;

[0062] Potassium chloride: Shanghai Aladdin Biotechnology Co., Ltd.;

[0063] Catalase: Shanghai Aladdin Biotechnology Co., Ltd.;

[0064] Potassium hydrogen phthalate: Shanghai Aladdin Biotechnology Co., Ltd.;

[0065] Sodium hydroxide: Shanghai Aladdin Biotechnology Co., Ltd.;

[0066] Luminol: Shanghai Aladdin Biotechnology Co., Ltd.;

[0067] Sodium dihydrogen phosphate hydrate: Shanghai Aladdin Biotechnology Co., Ltd.;

[0068] Disodium hydrogen phosphate hydrate: Shanghai Aladdin Biotechnology Co., Ltd.;

[0069] p-Benzoquinone: Shanghai Aladdin Biotechnology Co., Ltd.;

[0070] tert-Butanol: Shanghai Aladdin Biotechnology Co., Ltd.;

[0071] Uric acid: Shanghai Aladdin Biotechnology Co., Ltd.;

[0072] Platinum trichloride hydrate: Shanghai Aladdin Biotechnology Co., Ltd.;

[0073] TNF-α: Beijing Keyue Zhongkai Biotechnology Co., Ltd.;

[0074] IL-6: Beijing Keyue Zhongkai Biotechnology Co., Ltd.;

[0075] IL-1β: Sangon Biotech (Shanghai) Co., Ltd.;

[0076] PLGF: Sangon Biotech (Shanghai) Co., Ltd.

[0077] Ascorbic acid: Shanghai Maclean Biochemical Technology Co., Ltd.;

[0078] Aluminum nitrate nonahydrate: Shanghai Maclean Biochemical Technology Co., Ltd.;

[0079] Cobalt nitrate hexahydrate: Shanghai Maclean Biochemical Technology Co., Ltd.;

[0080] Chloroplatinic acid: Shanghai Maclean Biochemical Technology Co., Ltd.;

[0081] Urea: Tianjin Yongda Chemical Reagent Development Center;

[0082] Sodium chloride: Tianjin Yongda Chemical Reagent Development Center;

[0083] CRP: Nanjing Oukai Biotechnology Co., Ltd. (Nanjing, China);

[0084] NGAL: Nanjing Oukai Biotechnology Co., Ltd. (Nanjing, China);

[0085] Glucose: Comio Chemical Reagent Co., Ltd.;

[0086] Acetone: Hangzhou Shuanglin Chemical Reagent Co., Ltd.;

[0087] Anhydrous ethanol: Hangzhou Shuanglin Chemical Reagent Co., Ltd.;

[0088] Example 1

[0089] A layered CoAl double hydroxide (LDH) was grown on ITO and etched to enrich it with oxygen vacancies (LDH-Ov). PtNPs were then loaded onto this modified electrode, and an immunoelectrode was prepared by immobilizing IL-1β antibody on its outer surface. The process of detecting IL-1β in serum samples using electrochemiluminescence was then described.

[0090] Preparation of CoAl-LDH on ITO Electrode

[0091] ITO conductive glass with dimensions of 1 cm × 2 cm was ultrasonically cleaned in acetone, ethanol and ultrapure water for 30 min in sequence to remove surface oil and impurities; after cleaning, the surface moisture was dried with N2 and placed in an 80℃ oven for 12 h for later use.

[0092] CoAl layered bimetallic hydroxide (CoAl-LDH) was grown on an ITO substrate using a solvothermal direct growth method. The specific steps were as follows: 0.33 mM Co(NO3)2·6H2O, 0.11 mM Al(NO3)3·9H2O, 4.0 mM urea, and 1.0 mM NH4F were dissolved in 100 mL of deionized water and stirred in the dark for 1 h until fully dissolved to obtain a precursor solution for LDH growth. The pretreated ITO electrode was vertically immersed in the precursor solution to ensure that the electrode surface was completely submerged. The beaker was placed in an 80℃ constant temperature water bath and reacted for 7 h. After the reaction was completed, the electrode was removed, rinsed three times with ultrapure water to remove residual precursor on the surface, and dried with N2 to obtain an LDH / ITO electrode.

[0093] Preparation of Pt@LDH-Ov / ITO electrode

[0094] Alkaline etching enriched the LDH / ITO surface with oxygen vacancies (LDH-Ov / ITO). Specifically, the LDH / ITO electrode was immersed in 3 M NaOH solution for 30 min, then removed and rinsed repeatedly with ultrapure water five times to remove residual NaOH. It was then dried with N2 to obtain the LDH-Ov / ITO electrode. Subsequently, Pt nanoparticles were electrodeposited in situ onto the modified electrode at a constant voltage of -0.2 V for 2 s to prepare the Pt@LDH-Ov / ITO electrode. The same procedure was also used as a control for the electrodeposition of Pt nanoparticles on ITO and LDH / ITO. The electrodeposition solution mainly consisted of 0.5 mM H2PtCl6 solution (deionized water as the electrolyte), and the scan rate was 100 mV / s. After deposition, the electrode surface was gently rinsed with deionized water and dried with N2 to obtain the target electrode.

[0095] (3) Preparation of immunosensors and electrochemiluminescence detection of IL-1β

[0096] To further construct the immunorecognition interface, IL-1β antibody (10 μg / mL, 50 μL, phosphate buffer) was drop-coated onto a Pt@LDH-Ov / ITO electrode and incubated at 4°C for 60 min. The IL-1β antibody contains -NH2, and the Pt-NH2 bonds immobilized the antibody on the modified electrode surface, resulting in the Anti-IL-1β / Pt@LDH-Ov / ITO electrode. Subsequently, bovine serum albumin (BSA, 0.5%, w / w) was drop-coated onto the electrode and incubated at 4°C for 10 min to block non-specific sites, yielding a modified BSA / Anti-IL-1β / Pt@LDH-Ov / ITO electrode used as the working electrode for subsequent detection. During the construction of the immunosensor, rinsing with phosphate buffer (0.01 M, pH = 7.4) was performed between each step to remove unbound biomolecules from the surface.

[0097] Different concentrations of IL-1β (50 μL) were drop-coated onto the working electrode and incubated at 4 °C for 60 min. After incubation, the electrode surface was gently rinsed with phosphate buffer solution (0.01 M, pH = 7.4) to remove unbound target material. The resulting electrode was named the IL-1β / BSA / Anti-IL-1β / Pt@LDH-Ov / ITO electrode.

[0098] The detection solution was a phosphate buffer solution containing luminol (100 μM) (0.01 M, pH = 7.4). The detection potential was –1.0 ~ 0.8 V (scan rate 100 mV / s), and the photomultiplier tube voltage was set to 750 V.

[0099] The process of detecting serum IL-1β using the surface-immobilized IL-1β antibody-modified working electrode of Example 1 via electrochemiluminescence method is as follows: Serum was diluted 50-fold with phosphate buffer (0.01 mol / L, pH = 7.4), and a certain amount of IL-1β was added proportionally, resulting in IL-1β concentrations of 1 fg / mL, 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, and 100 ng / mL. IL-1β was drop-coated onto a BSA / Anti-IL-1β / Pt@LDH-Ov / ITO electrode and incubated at 4°C for 1 h. Unbound IL-1β was washed with phosphate buffer (0.01 mol / L, pH = 7.4) to obtain the test electrode. The electrode to be tested was placed in the electrochemiluminescence detection solution for electrochemiluminescence testing. A simple three-electrode system was used, with a silver / silver chloride electrode as the reference electrode (the solution was saturated potassium chloride), a platinum wire as the counter electrode, and a potential range of -1.0 ~ 0.8 V. (4) Standards and performance tests

[0100] I. Morphological observation and electrochemical characterization

[0101] Scanning electron microscopy images of the LDH / ITO electrode obtained in step (1) and the LDH-Ov / ITO electrode obtained in step (2) of Example 1 are shown below. Figure 1 As shown. Figure 1 Figure A shows a top view of the SEM at different magnifications. As can be seen, many large-sized two-dimensional LDH sheet-like structures are vertically arranged on the ITO substrate, with a smooth and intact surface. After alkaline etching (Figure B), the LDH sheet-like structures show obvious cracking, indicating that the introduction of oxygen vacancies leads to changes in the crystal structure; EDS and elemental mapping analysis (…) Figure 1 C- Figure 1 (D) The uniform distribution of Co, Al, and O elements was confirmed, indicating the successful synthesis of LDH. II. Characterization of Pt nanoparticles and EMSI verification: To further verify the structural element distribution and the successful deposition of PtNPs, XPS characterization was performed. Figure 2 These are XPS characterization data for different electrodes in Example 1. Figure 2 In the image, A represents XPS images of LDH / ITO, LDH-Ov / ITO, Pt@LDH / ITO, and Pt@LDH-Ov / ITO. Figure 2 In the image, B represents the high-resolution XPS deconvolutioned Co2p images in Pt@LDH / ITO and Pt@LDH-Ov / ITO. Figure 2C represents the high-resolution XPS deconvolutioned image of Pt@LDH / ITO and Pt@LDH-Ov / ITO at time 1s.

[0102] Figure 2 Figure A shows the total spectra of LDH / ITO, LDH-Ov / ITO, Pt@LDH / ITO, and Pt@LDH-Ov / ITO. The figure reveals peaks at C 1s, O 1s, Co 2p, and Al 2p, further confirming the successful synthesis of LDH. Furthermore, after PtNP deposition, corresponding peaks at the corresponding electron volts also appear, further demonstrating the successful deposition of PtNPs. To further investigate the EMSI interaction between PtNPs and LDH-Ov, XPS was also used to analyze the samples.

[0103] In addition, the Co 2p spectrum ( Figure 2 (B) shows the Co content in the Pt@LDH / ITO and Pt@LDH-Ov / ITO electrodes. 2+ There are two peaks at 781.3 eV and 797.2 eV, Co 3+ There are two peaks at 783.2 eV and 798.7 eV. The peaks at 786.6 eV and 802.9 eV are attributed to Sat., Co. 2+ and Co 3+ The coexistence of these is manifested in partial valence state changes. It is worth noting that Co... 3+ / Co 2+ The proportion of peaks gradually increased (from 32.96% to 67.53%), which is a necessary condition for EMSI to occur, indicating that Ov-Co 3+ The generation of sites.

[0104] Figure 2 The C-values ​​show that the O 1s spectra of the Pt@LDH / ITO and Pt@LDH-Ov / ITO electrodes split into three peaks at 532.6, 531.9, and 531.2 eV, which are attributed to adsorbed molecular water (OH), surface adsorbed oxygen (Oo) in LDH, and oxygen vacancies with low oxygen coordination (Ov), respectively. Furthermore, the oxygen vacancy peak content increased from 28.3% to 38.5% after alkaline etching, indicating that more Ov was generated.

[0105] Figure 3 The electrochemical performance of different electrodes in Example 1 is shown. Figure 3 In Figure A, the ECL response of different electrodes in the presence of luminol (100 μM) is shown. Figure 3 In the diagram, B represents the signal stability of different electrodes. Figure 3 C represents the stability and repeatability of the Pt@LDH-Ov / ITO electrode. Figure 3Figure D shows the ECL potential curves of each electrode obtained in PBS (0.01 M, pH = 7.4) containing 100 μM luminol. Figure 3 E represents the CV curves of Pt@LDH-Ov / ITO in PBS (0.01 M, pH 7.4) and luminol (100 μM) under different atmospheres (the inset shows the ECL signal of Pt@LDH-Ov / ITO under different atmospheres). Figure 3 F represents the ECL signal of Pt@LDH-Ov / ITO in different electrolytes. PBS (0.01 M, pH = 7.4) containing 100 μM luminol was supplemented with BQ (100 μM), TBA (100 μM), and CAT (100 μM), respectively. I and I 0 represents the ECL signal with or without ROS radical scavengers. The scan rate is 100 mV / s. Figure 3 G represents the ECL signal amplification strategy of Pt@LDH-Ov / ITO.

[0106] Comparison of ECL signals from different electrodes ( Figure 3 (A) shows that the signal intensity of the Pt@LDH-Ov / ITO electrode is significantly higher than that of other electrodes, being 12 times that of Pt@LDH / ITO. This confirms that the EMSI effect between LDH-Ov and PtNPs can significantly enhance the ECL signal, and that the signal is very stable. Figure 3 (Chinese BC). Figure 3 Figure D shows the ECL-potential curves for different electrodes. The figure indicates that all electrodes exhibit some catalytic activity towards luminol, with the Pt@LDH-Ov / ITO electrode showing the strongest catalytic effect. In addition to verifying the catalytic activity of luminol, CV scans and ECL signals were investigated using different electrodes under different atmospheres. Figure 3 In Figure 3 (E), it was found that the Pt@LDH-Ov / ITO electrode exhibited the highest current and strongest ECL signal under an O2 atmosphere, proving that the co-reactants in this invention are reactive oxygen species (ROS) generated by dissolved oxygen. Subsequent ROS scavenging experiments (Figure 3, F) showed that the superoxide anion radical scavenger p-benzoquinone reduced the signal by 82%, indicating that O2... ・- It is the main active species involved in luminol luminescence. Therefore, its reaction mechanism is inferred to be... Figure 3 As shown in G, depositing PtNPs on oxygen-vacancy-rich LDH can catalytically reduce dissolved oxygen to O2 to a greater extent. ·- This enhances its electrochemiluminescence signal.

[0107] To demonstrate the role of PtNPs and oxygen vacancies in enhancing the ECL signal, the electroactive area sizes of LDH-Ov / ITO, Pt@LDH / ITO, and Pt@LDH-Ov / ITO were measured. Figure 4 Different electrodes from Example 1 were used in a solution containing 2.5 mM (1:1) [Fe(CN)6]. 3- / 4- CV curves and linear relationships obtained at different scan rates in 0.1 M KCl. Figure 4 In the middle, A represents the CV curve of the LDH-Ov / ITO electrode. Figure 4 B represents the CV curve of the Pt@LDH / ITO electrode. Figure 4 C represents the CV curve of the Pt@LDH-Ov / ITO electrode. Figure 4 D in the figure represents the linear relationship between the LDH-Ov / ITO electrodes. Figure 4 E in the figure represents the linear relationship between the Pt@LDH / ITO electrode. Figure 4 The graph in Figure F shows the linear relationship between the Pt@LDH-Ov / ITO electrode. The scan rates used for the CV curves, from top to bottom, are 220, 200, 180, 160, 140, 120, 100, 80, 60, 40, and 20 mV / s. The linear relationship between peak current and the square root of the scan rate (DF) is also shown.

[0108] Figure 4 D- Figure 4 The presence of F indicates that the electrochemical reaction process occurring on the electrode surface is diffusion-controlled.

[0109] and Figure 4 China A and Figure 4 Compared to C, the electroactive area remained almost unchanged after Pt deposition, while the ECL signal increased several times, proving that the signal enhancement was due to the catalysis of Pt nanoparticles, rather than the increase in electroactive area. Figure 4 China B and Figure 4 Compared to C, the electroactive area of ​​the etched electrode is not much different, while the ECL signal is significantly improved, which further proves the generation of oxygen vacancies and their role in this invention.

[0110] Performance of the III electrochemiluminescence immunosensor for detecting IL-1β

[0111] To obtain the optimal experimental conditions, the etching time of LDH, the deposition time of PtNPs, the antibody incubation time of IL-1β, and the incubation time of IL-1β antigen were optimized. Figure 5This is the optimized ECL response of Pt@LDH-Ov / ITO in the presence of 100 μM luminol, considering different LDH etching times, PtNP deposition times, IL-1β antibody incubation times, and IL-1β antigen incubation times. Figure 5 In the figure, A represents different LDH etching times. Figure 5 In the middle, B represents the deposition time of different PtNPs. Figure 5 In the middle, C represents the incubation time of antibodies against different IL-1β. Figure 5 In the figure, D represents the incubation time for different IL-1β antigens. The IL-1β antibody concentration was 10 μg / mL. The IL-1β antigen concentration was 1 ng / mL. PMT = 750 V. The scan rate was 100 mV / s.

[0112] The etching time of LDH in step (3) of Example 1 was changed to 0, 10, 20, 30, 40, 50, and 60 min, respectively. Figure 5 As shown in Figure A, the optimal etching time is 30 minutes.

[0113] The deposition time of PtNPs in step (3) of Example 1 was changed to 0.5, 1, 2, 5, 8, and 10 s, respectively. Figure 5 As shown in Figure B, the optimal deposition time is 1 s.

[0114] The incubation time of the IL-1β antibody and the incubation time of the IL-1β antigen in step (3) of Example 1 were changed to 0, 10, 30, 60, 90, and 120 min, respectively. The test results are as follows: Figure 5 China B and Figure 5 As shown in Figure C, the preferred incubation time for both is 0.5-1 h, and the optimal incubation time is 1 h.

[0115] Figure 6 ECL signal, CV curve, and EIS curve of immunosensors fabricated with different electrodes in Example 1. Figure 6 In Figure A, ECL signals were obtained from different electrodes in PBS (0.01 M, pH = 7.4) containing 100 μM luminol. The concentration of IL-1β antibody was 10 μg / mL and the concentration of IL-1β antigen was 1 ng / mL. Figure 6 B represents the CV curves of immunosensors fabricated with different electrodes. Figure 6 C represents the EIS curves of immunosensors fabricated with different electrodes. The test solution contained 2.5 mM Fe(CN)6. 3- / 4- The 0.1 MKCl solution was scanned at a rate of 100 mV / s.

[0116] Different Pt@LDH-Ov / ITO electrodes (Pt@LDH-Ov / ITO, Anti-IL-1β / Pt@LDH-Ov / ITO, BSA / Anti-IL-1β / Pt@LDH-Ov / ITO, IL-1β / BSA / Anti-IL-1β / Pt@LDH-Ov / ITO) prepared in Example 1 were placed in a buffer system containing 100 μM luminol. Comparison of the ECL response revealed that the ECL intensity also decreased accordingly. Figure 6 (A). This may be attributed to the gradual modification of the electrodes, leading to increased steric hindrance, which in turn affects mass transfer, resulting in a gradual decrease in the ECL signal. This demonstrates that an immunosensor constructed based on Pt@LDH-Ov-ITO electrodes can detect IL-1β. Simultaneously, the CV curves of each electrode ( Figure 6 (B) and electrochemical impedance spectroscopy ( Figure 6 (C) indicates that with each step of electrode modification, the current value gradually decreases, and the electrochemical impedance spectroscopy... R The increasing semicircular diameter of the CT scan further confirms the successful construction of the immune sensor.

[0117] Under optimal experimental conditions, different concentrations of IL-1β were incubated on the BSA / Anti-IL-1β / Pt@LDH-Ov / ITO electrode, and then the electrode was placed in a PBS buffer solution containing 100 μM luminol for ECL signal detection to evaluate its detection performance. Figure 7 The calibration curves for the ECL signal, ECL intensity, and IL-1β logarithmic concentration of the Anti-IL-1β / Pt@LDH-Ov-ITO immunosensor in Example 1 are shown. Figure 7 In Figure A, the ECL signal of the immunosensor is shown at different concentrations of IL-1β. Figure 7 Figure B shows the calibration curve of ECL intensity and IL-1β logarithmic concentration of the immunosensor. The electrolyte solution is PBS (0.01 M, pH = 7.4) containing luminol (100 μM).

[0118] like Figure 7 As shown in Figure A, the ECL signal obtained by the prepared ECL immunosensor decreased with increasing IL-1β concentration. Within the concentration range of 1 fg / mL to 100 ng / mL, the ECL intensity showed a good linear relationship with the logarithmic concentration of IL-1β. Figure 7 (B) The linear regression equation is: I ECL = -953 (±23) logC IL-1β(pg / mL) + 6116 (±63), with a correlation coefficient of 0.996, the calculated limit of detection (LOD) is 0.47 fg / mL. Compared with other sensors for detecting IL-1β, our ECL immunosensor based on EMSI has a wider detection range and a lower limit of detection, and the electrode material is easy to prepare with fewer electrode construction steps.

[0119] To demonstrate the selectivity and anti-interference capabilities of this ECL immune sensor, Figure 8 To demonstrate the anti-interference performance and stability of the immune sensor in Example 1, Figure 8 In section A, the specificity is for different antigens, with CRP and PIGF concentrations at 10 ng / mL, and Glu and K... + NO3 - AA, Na + The concentration is 1 mM, Cl - The concentrations were 1 mM for IL-1β, 1 mM for IL-6, TNF-α, IFN-γ, and NGAL, and 1 ng / mL for all of them. Figure 8 In the middle B, the signal stability of the immunosensor is given, with an IL-1β concentration of 100 pg / mL. Figure 8 In the figure, C represents the repeatability of the immunosensor, the IL-1β concentration is 1.0 ng / mL, the detection solution is PBS (0.01M, pH = 7.4) containing 100 μM luminol, PMT = 750 V, and the error bars represent the standard deviation of three measurements performed under the same experimental conditions.

[0120] like Figure 8 As shown in Figure A, when IL-1β is absent in the analyte, the ECL signal of the sensor remains essentially unchanged. However, when the antibody is incubated with IL-1β or a mixture containing IL-1β, the ECL signal decreases significantly, with the decrease values ​​being nearly equal. This fully demonstrates that the prepared sensor has a specific recognition capability only for the target analyte IL-1β and is not easily affected by other coexisting substances, proving its high selectivity. Figure 8 As shown in Figure B, continuous scanning of the ECL signal of the IL-1β / BSA / Anti-IL-1β / Pt@LDH-Ov / ITO electrode revealed a highly stable signal with a relative standard deviation (RSD) of approximately 1.8%, demonstrating the stability of this immunosensor during detection. Furthermore, using the ECL response of the sensor incubating the antigen as the detection signal, the RSD for all five sensors was found to be 1.3%, indicating excellent reproducibility. Figure 8 (C)

Claims

1. An electrochemiluminescence immuno sensor for detecting interleukin-1 β, characterized by, A three-electrode system comprising a working electrode, a reference electrode and a counter electrode; The working electrode is a modified electrode of CoAl layered double hydroxide, which is loaded with platinum nanoparticles after alkaline etching, then fixed with interleukin-1β antibody and sealed with non-specific sites to obtain the working electrode; The immunosensor further comprises an electrolyte containing a luminol solution; The working electrode preparation process comprises: Step 1, modifying CoAl layered double hydroxide on the electrode surface to obtain a CoAl-LDH modified electrode, and obtaining an LDH-Ov modified electrode rich in oxygen vacancies through alkaline etching; Step 2, immersing the LDH-Ov modified electrode in a solution containing a platinum precursor to obtain a Pt@LDH-Ov modified electrode with platinum nanoparticles loaded on the surface through electrodeposition; Step 3, dropping and coating a buffer solution containing IL-1β antibody on the surface of the Pt@LDH-Ov modified electrode for incubation, and then sealing non-specific sites through incubation with an aqueous bovine serum albumin solution to obtain the working electrode.

2. The electrochemiluminescence immuno-sensor for detecting interleukin-1β according to claim 1, wherein, The step 1 of modifying CoAl layered double hydroxide on the electrode surface specifically comprises: immersing the electrode into a precursor solution containing cobalt nitrate, aluminum nitrate, urea and ammonium fluoride, and reacting at 60-100℃ for 6-8h, and then obtaining the CoAl-LDH modified electrode through cleaning and drying; The molar concentration of cobalt nitrate in the precursor solution is 0.1-0.8 mM, the molar concentration of aluminum nitrate is 0.05-0.5 mM, the molar concentration of urea is 1-6 mM, and the molar concentration of ammonium fluoride is 0.5-3 mM; And / or, the electrode comprises any one of an 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.

3. The electrochemiluminescence immunosensor for detecting interleukin-1β according to claim 1, wherein, The alkaline etching comprises: immersing the CoAl-LDH modified electrode in a NaOH solution for soaking, and then obtaining the LDH-Ov modified electrode through deionized water cleaning and nitrogen blowing drying after taking out; The concentration of the NaOH solution is 1-10 mol / L, the soaking is carried out at room temperature, the soaking time is 15-45 min, and the solvent of NaOH includes any one or more of ethanol, water and diethyl ether.

4. The electrochemiluminescence immuno-sensor for detecting interleukin-1 β according to claim 1, wherein, The solution containing a platinum precursor is an aqueous solution of chloroplatinic acid with a concentration of 0.3-0.7 mM; And / or, the constant voltage for electrodeposition in step 2 is-1.0~0 V, the electrodeposition time is 1~10 s, and the temperature is room temperature.

5. The electrochemiluminescence immuno-sensor for detecting interleukin-1 β according to claim 1, wherein, The molar concentration of the buffer solution containing IL-1β antibody is 1~20 μg / mL; the solvent used includes any one or more of sodium chloride-ethylenediaminetetraacetic acid-tris-hydroxymethyl aminomethane buffer, tris-hydroxymethyl aminomethane-ethylenediaminetetraacetic acid buffer; the incubation conditions are 0-5℃ for 0.5-2 h; And / or, the concentration of the aqueous bovine serum albumin solution is 0.2~2%(w / w), the incubation is carried out at 0-5℃, and the incubation time is 5-15 min.

6. The method for preparing an electrochemiluminescence immuno sensor for detecting interleukin-1 β according to any one of claims 1 to 5, characterized in that, The method comprises the steps of: Step 1, modifying CoAl layered double hydroxide on the electrode surface to obtain a CoAl-LDH modified electrode, and obtaining an LDH-Ov modified electrode rich in oxygen vacancies through alkaline etching; Step 2, immersing the LDH-Ov modified electrode into a solution containing platinum precursor to obtain a Pt@LDH-Ov modified electrode with platinum nanoparticles loaded on the surface by electrodeposition; Step 3, dropping and coating a buffer solution containing IL-1β antibody on the surface of the Pt@LDH-Ov modified electrode to incubate, and then incubating with bovine serum albumin aqueous solution to close non-specific sites to obtain the working electrode; Step 4, forming a three-electrode system with the working electrode, a counter electrode and a reference electrode to obtain the immunosensor, and using a solution containing luminol as an electrolyte when the immunosensor is detected.

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