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Exosome double-membrane protein co-expression detection platform based on magnetic separation and catalytic hairpin assembly as well as preparation method and application of exosome double-membrane protein co-expression detection platform

A magnetic separation and detection platform technology, applied in the biological field, can solve the problems of only detecting a single membrane protein, low detection sensitivity, and complicated operation, and achieve the effects of improving detection efficiency and diagnostic efficiency, high detection sensitivity, and improving detection sensitivity

Active Publication Date: 2020-04-10
NANFANG HOSPITAL OF SOUTHERN MEDICAL UNIV
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Problems solved by technology

[0004] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide an exosome double-membrane protein co-expression detection platform based on magnetic separation and catalytic hairpin assembly and its preparation method and application, which are used to solve the problems of the prior art. Exosome membrane protein detection methods have problems such as low detection sensitivity, long time-consuming, complicated operation, and only a single membrane protein can be detected

Method used

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  • Exosome double-membrane protein co-expression detection platform based on magnetic separation and catalytic hairpin assembly as well as preparation method and application of exosome double-membrane protein co-expression detection platform
  • Exosome double-membrane protein co-expression detection platform based on magnetic separation and catalytic hairpin assembly as well as preparation method and application of exosome double-membrane protein co-expression detection platform
  • Exosome double-membrane protein co-expression detection platform based on magnetic separation and catalytic hairpin assembly as well as preparation method and application of exosome double-membrane protein co-expression detection platform

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preparation example Construction

[0047] The invention provides an exosome double-membrane protein co-expression detection platform based on magnetic separation and catalytic hairpin assembly, as well as its preparation method and application. figure 1 It is shown as the detection principle diagram of the present invention.

[0048] Such as figure 1 As shown, in this detection platform, magnetic beads are coated with antibody CD63, and after incubation with exosomes, exosomes are separated and enriched by magnetic separation, and then Apt EpCAM -T, as the recognition element and catalytic reaction initiation element of exosomes, is jointly constructed with the hairpin structure H1 and the hairpin structure H2 with FAM as the fluorescent reporter group and BHQ1 as the fluorescent quencher group. This detection platform can realize the qualitative detection of double-membrane protein co-expression in exosomes.

[0049] Among them, the sequence of the issuing structure H1 is:

[0050] FAM-AACCCTAACCCTAAACCCTCC...

Embodiment 1

[0057] Feasibility analysis of an exosomal double-membrane protein detection platform based on magnetic separation method enrichment and catalytic hairpin assembly (CHA)

[0058] 1. Verify the expression of CD63 protein on the exosome membrane by super-resolution microscopy imaging and western blot experiments

[0059] The co-localization of CD63 protein with exosome membranes was characterized by super-resolution imaging to verify the expression of CD63 on cell line-derived exosome membranes.

[0060] (1) The experimental method of super-resolution imaging technology is as follows:

[0061] 1) PLL-coated coverslip (poly-lysine-coated slides), covered with 50 μl 1mg / ml Poly-L-lysine on the shooting dish, incubated at room temperature for 30 minutes, and washed 3 times with appropriate amount of PBS.

[0062] 2) In 10 μL sample + 40 μL PBS (set different concentrations), add 50 μL to the photographing dish, and incubate at room temperature for 30 min.

[0063] 3) Add 50 μL of...

Embodiment 2

[0100] Feasibility verification of an exosomal double-membrane protein detection platform based on magnetic separation and catalytic hairpin assembly (CHA)

[0101] In order to verify the feasibility of the exosome double membrane protein detection platform based on magnetic separation method and CHA, we conducted the following experiments:

[0102] 1) Pipette 50ul of magnetic beads coated with CD63 antibody into a 1.5ml PE tube, place on a magnetic stand for magnetic separation, and keep the precipitated magnetic beads.

[0103] 2) Wash the magnetic beads once with 200ul 0.1% BSA PBS buffer solution. After each washing, place the sample on the magnetic stand to allow it to fully settle (put the PE tube on its side for 30 seconds, then stand it upright for 60 seconds), collect the precipitate, and finally Resuspend the pellet with 200ul 0.1% BSAPBS buffer.

[0104] 3) Add 5ul exosomes (4.01×10 11 / mL), incubated on a shaker for 18-22h.

[0105] 4) The sample is placed on a ...

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Abstract

The invention belongs to the technical field of biology and particularly discloses an exosome double-membrane protein co-expression detection platform based on magnetic separation and catalytic hairpin assembly as well as a preparation method and application thereof. The preparation method comprises the following steps: co-incubating an exosome and a magnetic bead coated with an antibody CD63; separating and enriching exosome through a magnetic separation method with AptEpCAM-T as acting as a recognition element and a catalytic reaction starting element of the exosome, then enabling a hairpinstructure H1 and a hairpin structure H2 jointly to construct a detection platform to achieve qualitative detection of exosome double-membrane protein co-expression. By adopting a magnetic separation method, the interference of free or impurity proteins is effectively eliminated, and the detection accuracy and specificity are improved; a single target protein is converted into a large number of detectable fluorescence signals by adopting a catalytic hairpin assembly technology, so that the detection sensitivity is improved, and meanwhile, the design of exosome double-membrane protein detectionalso improves the detection efficiency and the diagnosis efficiency.

Description

technical field [0001] The present invention relates to the field of biotechnology, and relates to an exosome membrane protein detection technology, in particular to an exosome double membrane protein co-expression detection platform based on magnetic separation and catalytic hairpin assembly and its preparation method and application. Two membrane proteins in exosomes were simultaneously detected by an ultra-centrifugal fluorescence method. Background technique [0002] Exosomes refer to small membrane vesicles (30-150nm) that contain complex RNA and proteins. Today, they specifically refer to discoid vesicles with a diameter of 40-100nm. Exosomes can carry proteins, transport RNA, and play an important role in intercellular material and information transduction; exosomes may regulate immune function, promote tumor angiogenesis and tumor metastasis, and directly act on tumor cells, etc. Tumor progression. Membrane proteins carried by exosomes are considered to be biomarke...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/53
CPCG01N33/68G01N33/53G01N2333/70596G01N2333/705
Inventor 郑磊陈贤华潘炜伦李博张晔罗世华司徒博
Owner NANFANG HOSPITAL OF SOUTHERN MEDICAL UNIV
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