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Cytokine multiple detection method based on dual coding and monomolecular counting

A single-molecule counting and cytokine technology, applied in measurement devices, biological tests, analytical materials, etc., can solve the problems of unsatisfactory sensitivity, increased detection complexity, low content, etc., and achieve the effect of great application potential.

Active Publication Date: 2016-02-24
SHANDONG UNIV
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Problems solved by technology

However, due to the extremely low content of such cancer markers in serum, their sensitivity still cannot meet the needs of actual detection.
In addition, this method needs to release the barcode strands from the nanoparticles, which increases the complexity of the detection

Method used

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  • Cytokine multiple detection method based on dual coding and monomolecular counting
  • Cytokine multiple detection method based on dual coding and monomolecular counting
  • Cytokine multiple detection method based on dual coding and monomolecular counting

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Embodiment Construction

[0022] Reagents and materials Human tumor necrosis factor alpha (TNF-α), recombinant human interferon gamma (IFN-γ), tumor necrosis factor alpha antibody (anti-TNF-α), biotinylated tumor necrosis factor alpha antibody (bio-anti -TNF-α), interferon γ antibody (anti-IFN-γ), biotinylated interferon γ antibody (bio-anti-IFN-γ) and human IgG were purchased from Abcam. Streptavidin-MNBs (350 nm) were purchased from Bangs Laboratories. Bovine serum albumin (BSA) was purchased from Shanghai Sangon Bioengineering Co., Ltd. (Shanghai). All the solutions used in the experiment were prepared with ultrapure water, and all the nucleic acid sequences were synthesized by Yingwei Jieji Co., Ltd. (Beijing).

[0023] Instrument Epi-fluorescence microscope system (OlympusOpticalCo.Ltd, Tokyo, Japan), mainly including inverted microscope (OlympusIX81), halogen lamp (OlympusLG-PS2), inverted fluorescence microscope control unit (OlympusIX2-UCB), electronic multi-stage amplification inductive coupl...

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Abstract

The invention discloses a cytokine multiple detection method based on dual coding and monomolecular counting. The method is characterized in that a first antibody is fixed on a same glass substrate, through interaction of antigen-antibodies, the target objects can be respectively fixed; simultaneously, a secondary antibody and a first coding strand are modified on magnetic nano-particles to obtain a magnetic nano probe; then specific binding effect between the secondary antibody and antigen is used, the magnetic nano probe is fixed on a substrate to form an immune complex having a sandwich structure, the first coding strand on the magnetic nano probe is taken as an amplification unit to trigger a multi-branches hybridization chain reaction, and a double-chain structure having multiple branches; the branched chain is a secondary coding strand; finally, the secondary coding strand and a polymolecule-labeled fluorescence probe are combined, an enhanced fluorescence signal is generated, and quantification on the target object is carried out by counting the amount of phosphor dots. The method realizes multiple and sensitive detection for cytokine, and a detection limit of two target objects is 5fM.

Description

technical field [0001] The invention relates to a method for multiple detection of cytokines based on double coding and single molecule counting. Background technique [0002] Cytokines are a class of low-molecular-weight proteins secreted by immune cells, which usually play a role in signal transduction in immune responses. Recently, studies have found that some cytokine-mediated signaling pathways are related to the survival, invasion and metastasis of tumor cells, so these cytokines have been used as biomarkers for cancer diagnosis and treatment. However, since a cancer may have multiple markers or a marker may correspond to multiple cancers, it is more accurate and feasible to use multiple biomarkers to detect cancer. [0003] The commonly used cytokine detection method is enzyme-linked immunosorbent assay (ELISA), which needs to first immobilize cytokines on specific antibodies, and then use enzyme or fluorophore-labeled secondary antibodies to detect them. However, d...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/533
CPCG01N33/533G01N33/68
Inventor 王磊姜玮李伟
Owner SHANDONG UNIV
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