Super-paramagnetic nanoparticle for capturing exosomes and preparation method thereof as well as specific exosome luminescence immune quantitative detection kit

A nanoparticle, luminescent immune technology, applied in biological testing, measuring devices, material testing products, etc., can solve the problems of inconvenience, time-consuming, and unfavorable clinical routine development, and achieve simple operation, high sensitivity, and short detection time. Effect

Inactive Publication Date: 2018-08-10
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the detection of specific exosomes in body fluids is mainly through the separation and purification of exosomes by ultracentrifugati

Method used

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  • Super-paramagnetic nanoparticle for capturing exosomes and preparation method thereof as well as specific exosome luminescence immune quantitative detection kit
  • Super-paramagnetic nanoparticle for capturing exosomes and preparation method thereof as well as specific exosome luminescence immune quantitative detection kit
  • Super-paramagnetic nanoparticle for capturing exosomes and preparation method thereof as well as specific exosome luminescence immune quantitative detection kit

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Experimental program
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Example Embodiment

[0028] The present invention also provides a method for preparing the superparamagnetic nanoparticles for capturing exosomes, including the following steps: 1) The coupling unit, iron oxide nanoparticles and chloroform are subjected to an oxidation reaction under ultrasonic conditions to obtain The carboxyl modified iron oxide nanoparticles are a superparamagnetic nanoparticle matrix; 2) the superparamagnetic nanoparticle matrix is ​​condensed and coupled with an exosome shared marker antibody to obtain the superparamagnetic nanoparticle that captures the exosomes.

[0029] In the present invention, the carboxyl phospholipid polyethylene glycol is used to oxidize the iron oxide nanoparticles to obtain the carboxyl modified iron oxide nanoparticles, which is the superparamagnetic nanoparticle matrix. In the present invention, the particle size of the iron oxide nanoparticles is preferably 2-40 nm; the preparation method of the iron oxide nanoparticles preferably includes: mixing ir...

Example Embodiment

[0069] Example 1

[0070] Confirmation of common and specific markers of exosomes

[0071] Cultivate pancreatic cancer cell line Panc-1 and normal pancreatic cell line HPDE with RPMI 1640 containing 10% fetal bovine serum (FBS). When the growth density reaches 80%, replace the RPMI 1640 medium without FBS and continue to culture for 36 hours; collect and culture The supernatant is used to extract exosomes. Dispense the supernatant into 50mL centrifuge tubes, centrifuge at 1000g for 10min to remove cell debris; centrifuge the supernatant at 10,000g for 20min to remove microvesicles and cell debris; take the supernatant and centrifuge at 110,000g for 70min, remove the supernatant and resuspend the pellet in PBS buffer After the substance was centrifuged again at 110,000g for 70 min, a precipitate (exosomes) was obtained. Resuspend the pellet with 100μL PBS buffer for subsequent experiments: transmission electron microscopy observation and identification of the shape of the extracte...

Example Embodiment

[0072] Example 2

[0073] Screening of antibodies specifically binding to exosomal markers

[0074] Using flow cytometry technology to identify self-made antibodies that can bind to specific exosomal surface protein markers include GPC1, GPC3, PSMA, TMEM256 and EpCAM. take Exocytosis PBS, add Aldehyde / sulfate beads (aldehyde / sulfate beads), mix well at room temperature for 15 minutes. Join PBS, 4°C overnight. Join 1M glycine, mix at room temperature for 30 min. Centrifuge at 12000rpm for 1.5min. Remove the supernatant and use Resuspend the pellet in 10% BSA. Mix and seal at room temperature for 1 hour. Centrifuge at 12,000 rpm for 1.5 min. Add separately after removing the supernatant GPC3, PSMA, TMEM256 or EpCAM, etc., the corresponding antibody is diluted with 2% BSA to Mix at room temperature for 1 hour. Join 2% BSA, centrifuge at 12,000 rpm for 1.5 min, remove the supernatant, and add After centrifugation with 2% BSA at 12,000 rpm for 1.5 min, the supernatant ...

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Abstract

The invention provides a super-paramagnetic nanoparticle for capturing exosomes and a preparation method thereof as well as a specific exosome luminescence immune quantitative detection kit, and belongs to the technical field of exosome detection. The super-paramagnetic nanoparticle for capturing the exosomes, provided by the invention, comprises a super-paramagnetic nanoparticle base body and anexosome shared marker antibody coupled with the super-paramagnetic nanoparticle base body. The specific exosome luminescence immune quantitative detection kit provided by the invention comprises the super-paramagnetic nanoparticle for capturing the exosomes, an exosome specific marker antibody for luminescence labeling, confining liquid, a washing solution, a NaOH water solution, hydrogen peroxideand a calibrator, and can realize simple, rapid and quantitative detection of the specific exosomes; the specific exosome luminescence immune quantitative detection kit can be directly used for detecting a marker of the specific exosomes in blood serum, blood plasma, pleuroperitoneal fluid, urine and cerebrospinal fluid samples; the specific exosome luminescence immune quantitative detection kithas the advantages of high sensitivity, good stability, short detection time and simplicity in operation and is very applicable to clinical detection.

Description

technical field [0001] The invention belongs to the technical field of exosome detection, and in particular relates to a superparamagnetic nanoparticle for capturing exosomes, a preparation method thereof, and a specific exosome luminescent immunoquantitative detection kit. Background technique [0002] Exosomes are double-membrane vesicles with a size of 30nm to 200nm released by cells to the outside of the cell, carrying a large amount of proteins, lipids, and RNAs, and mediating information transmission between cells in the body, thereby affecting the physiological functions of cells. In addition to common markers (such as CD9, CD63, CD81, etc.), the surface of the exosome membrane also expresses a variety of specific markers (such as GPC1, GPC3, PSMA, TMEM256, EpCAM, etc.), and these specific exosomes highly reflect host cell type and state. Studies have shown that the isolation and detection of specific exosomes is of great significance for disease diagnosis, treatment...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/543
CPCG01N33/68G01N33/54326G01N33/54346
Inventor 陶志华
Owner ZHEJIANG UNIV
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