CN-122017225-A - Electrochemiluminescence immunosensor for detecting interleukin-1 beta, preparation method and application thereof

Abstract

The invention relates to the technical field of electrochemiluminescence sensing, and discloses an electrochemiluminescence immunosensor for detecting interleukin-1 beta, a preparation method and application thereof, wherein the electrochemiluminescence immunosensor comprises a working electrode, a reference electrode and a counter electrode, the working electrode comprises a base electrode and an ordered mesoporous silica film (SNF) modified on the surface of the base electrode, a composite nano-enzyme containing cobaltosic oxide nano-particles and nitrogen doped graphene quantum dots is loaded in a nano pore canal of the SNF, an epoxy group is modified on the outer surface of the SNF, an anti-interleukin-1 beta antibody is covalently fixed through the epoxy group, and a detection system of the electrochemiluminescence immunosensor is a luminol/dissolved oxygen system. The electrochemiluminescence immunosensor can remarkably enhance luminol/dissolved oxygen system signals and realize high-sensitivity and accurate quantitative detection of interleukin-1 beta under neutral conditions.

Inventors

• ZHANG WEI
• MA XINYING
• WEI PU
• LIU JIYANG

Assignees

• 杭州市第一人民医院(西湖大学附属杭州市第一人民医院)

Dates

Publication Date

20260512

Application Date

20260416

Claims (10)

1. 1. An electrochemiluminescence immunosensor for detecting interleukin-1 beta, comprising a working electrode, a reference electrode, and a counter electrode; the working electrode comprises a substrate electrode and SNF modified on the surface of the substrate electrode; The nanometer pore canal of the SNF is internally loaded with a composite nanometer enzyme containing Co 3 O 4 nanometer particles and NGQDs, the outer surface of the SNF is modified with an epoxy group, and an anti-interleukin-1 beta antibody is covalently immobilized through the epoxy group; The detection system of the electrochemiluminescence immunosensor is a luminol/dissolved oxygen system.
2. 2. An electrochemiluminescence immunosensor for detecting interleukin-1 β according to claim 1, wherein the Co 3 O 4 nanoparticle is spaced no more than 3 nm from adjacent NGQDs in the nanopore.
3. 3. An electrochemiluminescence immunosensor for detecting interleukin-1 beta according to any one of claims 1-2, wherein the pH of the detection system is 6.8-7.6.
4. 4. A method for preparing an electrochemiluminescence immunosensor for detecting interleukin-1 beta, comprising the steps of: (1) SNF is modified on the surface of a substrate electrode to obtain an SNF modified electrode, and the SNF modified electrode is immersed into a silane solution containing epoxy groups for reaction to obtain an O-SNF modified electrode with the outer surface modified with the epoxy groups; (2) Immersing the O-SNF modified electrode into a solution containing CoSO 4 for electrodeposition to obtain a Co 3 O 4 @O-SNF modified electrode; (3) Immersing the Co 3 O 4 @O-SNF modified electrode into a solution containing NGQDs for electrophoresis treatment to obtain a NGQDs/Co 3 O 4 @O-SNF modified electrode; (4) Incubating NGQDs/Co 3 O 4 @O-SNF modified electrode with a buffer solution containing anti-interleukin-1 beta antibody, and blocking non-specific sites by using bovine serum albumin solution to obtain the working electrode.
5. 5. The method for preparing an electrochemiluminescence immunosensor for detecting interleukin-1 beta according to claim 4, wherein in the step (2), the concentration of CoSO 4 in the solution containing CoSO 4 is 0.02-0.2 mol/L, the voltage of electrodeposition is +1.0 to +2.0V, and the deposition time is 1-20 s.
6. 6. The method for preparing an electrochemical luminescence immunosensor for detecting interleukin-1 beta according to claim 4, wherein in the step (3), the potential of the electrophoresis treatment is +0.4 to +1.0V, and the electrophoresis time is 30 to 180 s.
7. 7. The method for preparing an electrochemical luminescence immunosensor for detecting interleukin-1 beta according to claim 4, wherein in the step (1), the concentration of the epoxy group-containing silane in the solution is 2.0-3.0 mmol/L.
8. 8. The method for preparing an electrochemical luminescence immunosensor for detecting interleukin-1 beta according to claim 4, wherein in the step (4), the incubation temperature is 0-5 ℃ and the incubation time is 0.5-3 h.
9. 9. The use of an electrochemiluminescence immunosensor for detecting interleukin-1 beta for non-diagnostic purposes, which is characterized by comprising the steps of incubating a working electrode of the electrochemiluminescence immunosensor with a liquid to be detected containing interleukin-1 beta, placing the incubated working electrode, a reference electrode and a counter electrode in a luminol/dissolved oxygen system detection liquid, taking dissolved oxygen as a co-reactant, applying a scanning voltage, and quantitatively analyzing the interleukin-1 beta by detecting the reduction degree of an electrochemiluminescence signal.
10. 10. The use of an electrochemiluminescence immunosensor according to claim 9 for detection of non-diagnostic purpose interleukin-1 beta, wherein the concentration of luminol in the luminol/dissolved oxygen system detection solution is 10-200 μmol/L.

Description

Electrochemiluminescence immunosensor for detecting interleukin-1 beta, preparation method and application thereof Technical Field The invention relates to the technical field of electrochemiluminescence sensing, in particular to an electrochemiluminescence immunosensor for detecting interleukin-1 beta, a preparation method and application thereof. Background Interleukin-1 beta (IL-1 beta) is a core pro-inflammatory cytokine, and in the obstetrical field, the dynamic change of the level is a key biomarker for measuring the immunological status and the inflammatory balance of maternal-fetal interface, and has important prompt significance for evaluating serious pregnancy complications. The currently widely used interleukin-1 beta detection methods are enzyme-linked immunosorbent assay, colorimetry, chromatography and the like, but the pretreatment of the samples of the detection methods is complicated and expensive large-scale instruments are needed. The electrochemical luminescence (ECL) analysis technology is an analysis method for exciting luminescence reaction in an electrochemical mode, has the remarkable advantages of low background signal, simplicity and convenience in operation, wide dynamic range, strong time-space controllability and the like, and becomes a common analysis tool in the fields of biosensing, environment monitoring, clinical diagnosis and the like. In electrochemiluminescence analysis technology, ECL systems of luminol/coreactant systems are receiving extensive attention from researchers due to their low excitation potential and wide analytical applications. Since the system is still limited by low ECL luminescence quantum yield and insufficient signal intensity, the ECL intensity of the luminol/coreactant system is critical in improving the conventional electrode. The Chinese patent application with publication number of CN119086677A discloses an electrochemiluminescence immunosensor sensitized by nano-enzyme, a construction method and application thereof, wherein a silicon dioxide nano-pore membrane of nano-channel limited Co 3O4 nano-enzyme is used for modifying an electrode, and Co 3O4 nano-enzyme performs double electro-catalysis on luminol and hydrogen peroxide to enhance a luminol/hydrogen peroxide system signal, wherein the hydrogen peroxide is an exogenous Co-reactant. However, the scheme has the following limitations that firstly, the system relies on exogenous addition of unstable hydrogen peroxide to increase the complexity and background noise of the system, secondly, the catalytic efficiency is limited by the activity of single nano enzyme, the catalytic efficiency is limited, and furthermore, the system usually works under alkaline conditions and is incompatible with the neutral physiological environment of a biological sample, and the pH value of the sample to be measured needs to be additionally adjusted. In addition, the luminol/dissolved oxygen system adopting the endogenous coreactant can avoid the introduction of exogenous hydrogen peroxide, but the system has weak signal and low detection sensitivity under neutral conditions. In conclusion, the conventional electrochemiluminescence detection system is difficult to realize quantitative detection of interleukin-1 beta with simple operation, high sensitivity and accuracy. Disclosure of Invention In order to solve the technical problems, the invention provides an electrochemiluminescence immunosensor for detecting interleukin-1 beta, which can obviously enhance luminol/dissolved oxygen system signals and realize high-sensitivity and accurate quantitative detection of the interleukin-1 beta under neutral conditions. The first specific technical scheme of the invention is that the electrochemiluminescence immunosensor for detecting interleukin-1 beta comprises a working electrode, a reference electrode and a counter electrode; The working electrode comprises a substrate electrode and an ordered mesoporous silica film (SNF) modified on the surface of the substrate electrode; Composite nano enzyme containing cobaltosic oxide (Co 3O4) nano particles and nitrogen doped graphene quantum dots (NGQDs) is loaded in a nano pore canal of the SNF, an epoxy group is modified on the outer surface of the SNF, and an anti-interleukin-1 beta antibody is covalently immobilized through the epoxy group; The detection system of the electrochemiluminescence immunosensor is a luminol/dissolved oxygen system. According to the invention, the Co 3O4/NGQDs composite nano enzyme is constructed in the SNF nano reactor, the Co 3O4 and NGQDs form a tightly coupled synergistic catalytic interface by virtue of the finite field effect of SNF, and the Co 3O4 and NGQDs generate a remarkable synergistic catalytic effect, so that compared with single Co 3O4 nano enzyme or NGQDs component, the catalytic efficiency of a luminol/dissolved oxygen system is greatly improved, the luminol/dissolved oxygen system signal is remarkably enhanced, the luminol can rea