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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 ultracentrifugation, and then the detection of special markers of exosomes. This method is time-consuming, inconvenient, and not conducive to routine clinical practice.

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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preparation example Construction

[0028] The present invention also provides a method for preparing the superparamagnetic nanoparticles for capturing exosomes, which includes the following steps: 1) oxidizing the coupling unit, iron oxide nanoparticles and chloroform under ultrasonic conditions to obtain The carboxyl-modified iron oxide nanoparticles are a superparamagnetic nanoparticle matrix; 2) condensation coupling the superparamagnetic nanoparticle matrix with an exosome common marker antibody to obtain superparamagnetic nanoparticles capturing exosomes.

[0029] In the present invention, carboxyl phospholipid polyethylene glycol and iron oxide nanoparticles are oxidized to obtain 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 and stirring iron, oleic acid and octadecene at 300-3...

Embodiment 1

[0070] Consensus and specific marker confirmation of exosomes

[0071] Pancreatic cancer cell line Panc-1 and pancreatic normal cell line HPDE were cultured with RPMI 1640 containing 10% fetal bovine serum (FBS), and when the growth density reached 80%, the RPMI 1640 medium without FBS was replaced to continue culturing for 36 hours; The supernatant was used to extract exosomes. Dispense the supernatant into 50mL centrifuge tubes, centrifuge at 1000g for 10min to remove cell debris; continue to centrifuge the supernatant at 10000g for 20min to remove microvesicles and cell debris; take the supernatant and centrifuge at 110000g for 70min, remove the supernatant and resuspend the pellet with PBS buffer After the substance was centrifuged again at 110,000 g for 70 min, the precipitate (exosome) was obtained. The pellet was resuspended in 100 μL PBS buffer for subsequent experiments: the morphology of the extracted exosomes was identified by transmission electron microscopy; the ...

Embodiment 2

[0073] Screening of specific binding antibodies for exosome markers

[0074] Self-made antibodies that can bind to specific exosome surface protein markers identified by flow cytometry include GPC1, GPC3, PSMA, TMEM256 and EpCAM. Pick exosomes suspended in PBS, join Aldehyde / sulfate beads, mix at room temperature for 15 minutes. join in PBS, overnight at 4°C. join in 1M glycine, mix at room temperature for 30min. Centrifuge at 12000rpm for 1.5min. remove the supernatant, use Resuspend the pellet in 10% BSA. Mix well at room temperature and block for 1 hour. Centrifuge at 12,000 rpm for 1.5 min. After removing the supernatant, add GPC3, PSMA, TMEM256 or EpCAM, etc., the corresponding antibody was diluted with 2% BSA Mix well at room temperature for 1 hour. join in 2% BSA, centrifuged at 12,000rpm for 1.5min, removed the supernatant, added 2% BSA, centrifuge at 12,000rpm for 1.5min, then discard the supernatant. join in fluorescent secondary antibo...

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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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