Luminol and trisruthenium-based potential-resolved electrochemiluminescence detection method and application thereof

A bipyridyl ruthenium and luminescence detection technology, which is applied in the field of biomedicine, can solve problems such as difficulty in luminescence, and achieve the effect of eliminating mutual interference

Active Publication Date: 2014-06-25
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Purpose of the invention: In order to solve the technical problems existing in the prior art, the present invention proposes a potential-resolved electrochemiluminescence detection method based on luminol and bipyridylruthenium and its application, so as to solve the problem of simultaneous detection of two electrochemiluminescent substances. It is difficult to distinguish the luminescence of the two when they exist in one system. At the same time, this method is applied to the simultaneous detection of two antigens on the cell surface

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  • Luminol and trisruthenium-based potential-resolved electrochemiluminescence detection method and application thereof

Examples

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Embodiment 1

[0040] Example 1 Potential-resolved electrochemiluminescence of luminol and bipyridylruthenium in solution.

[0041] (1) Cross-reaction of ruthenium bipyridyl and luminol in solution at negative potential.

[0042] Detection conditions: when there are 3mM persulfate particles in the system, the luminescence curves of 100 μM bipyridyl ruthenium, a mixed solution of 100 μM bipyridyl ruthenium particles and 100 μM luminol, and 100 μM luminol are respectively detected under negative potential conditions. Among them, the whole detection system is in the PBS buffer solution of pH 7.4, an O-ring is attached to the surface of the ITO electrode as the solution chamber, the ITO electrode is used as the working electrode for luminescence detection, and the silver-silver chloride electrode is used as the reference electrode. The platinum electrode was used as the counter electrode, and the voltage sweep rate was -0.2V / s (the voltage sweep rate in the following embodiments all used -0.2V / s...

Embodiment 2

[0056] Example 2 Potential-resolved electrochemiluminescence of luminol and ruthenium bipyridyl on particle surface.

[0057] In order to simulate the simultaneous detection of two antigens on the cell surface using luminol and ruthenium bipyridyl potential-resolved electrochemiluminescence, we modified the antigen on a silica surface with a diameter of about 20 μm, which is similar to the cell size. In the analytical experiments, the particles settled on the electrode surface, simulating the adhesion behavior of cells. Specifically, include the following steps:

[0058] (1) Preparation of antibody-modified luminol complex: the preparation process is as follows Figure 5 (1) shown. Set the concentration to 10 -3 M's 3-mercaptopropionic acid (3-MPA) was first added to a solution of gold nanoparticles (Au NPs) with an average diameter of 13 nm. The mixed solution was reacted at 37°C for 12 hours, and then centrifuged at 14,000 rpm for 30 minutes to collect gold nanoparticle ...

Embodiment 3

[0071] Example 3 Combined detection of two antigens on the cell surface.

[0072] MCF-7 cells and PC-3 cells were cultured in DMEM high glucose medium and F- Cultured in 12K medium. Cells were cultured in 5% (v / v) CO 2 , in a cell culture incubator with a temperature of 37 °C and a humid atmosphere. Wherein, the antibiotic is a mixture of penicillin and streptomycin, wherein the concentration of penicillin is 100 U / ml, and the concentration of streptomycin is 100 μg / ml. That is, the total volume of the culture medium is 500ml, wherein the fetal bovine serum is 50ml, and the mixed solution of penicillin and streptomycin purchased as 10000U / ml and 10000μg / ml (purchased from Thermo Scientific's penicillin-streptomycin 100X solution (HyClone TM Penicillin-Streptomycin100X Solution), add 5ml into 500ml, which is equivalent to diluting 100 times to reach the concentration of penicillin 100U / ml and streptomycin 100μg / ml.

[0073] Such as Figure 10 As shown, the same process as...

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Abstract

The invention discloses a luminol and trisruthenium-based potential-resolved electrochemiluminescence detection method. Luminal and trisruthenium are used as luminescent probes. During detection, an ITO (indium tin oxide) electrode is used as a working electrode for luminescence detection, a silver-silver chloride electrode is used as a reference electrode, a potassium electrode is used as a counter electrode, and a luminescent signal from luminol is detected by performing scanning from 0.6V to 0.0V; peroxydisulfate and an excessive amount of luminol are added into a detection system, and a luminescent signal from trisruthenium is detected by performing scanning from 0.0V to -1.0V. The invention also discloses a simple and effective method for simultaneously detecting two cell surface cancer biomarkers. According to the methods, two antigens on the surfaces of cells can be analytically detected by one step by double-tagging and potential resolving methods, the mutual interference of signals under different potentials is eliminated, and carcinoembryonic antigens and alpha fetoprotein antigens on surfaces of cells are successfully detected at the same time.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a potential-resolving electrochemiluminescence detection method based on luminol and bipyridylruthenium and its application. Background technique [0002] Electrochemiluminescence (ECL) is another method applied to immunoassays that utilizes ECL probes instead of fluorophores. The electrochemiluminescence method does not require excitation by a light source, so it has the advantages of low background and simple equipment. The most commonly used ECL probes are luminol and ruthenium bipyridyl. In general, when a positive potential is applied, the luminol anion undergoes a one-electron oxidation process and becomes diazaquinone, which is further oxidized to 3-aminophthalic acid in an excited state, thereby returning to the ground state to emit light. For bipyridyl ruthenium probes, S 2 o 8 2- Ions, as co-reactants, are first reduced to SO at a negative potential...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/76G01N33/574
Inventor 方丹君江德臣姜晖韩芳霏
Owner NANJING MEDICAL UNIV
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