CN117330481B - Flow detection method for exosomes and application thereof - Google Patents
Flow detection method for exosomes and application thereof Download PDFInfo
- Publication number
- CN117330481B CN117330481B CN202311596892.1A CN202311596892A CN117330481B CN 117330481 B CN117330481 B CN 117330481B CN 202311596892 A CN202311596892 A CN 202311596892A CN 117330481 B CN117330481 B CN 117330481B
- Authority
- CN
- China
- Prior art keywords
- flow
- exosomes
- detection
- exosome
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 210000001808 exosome Anatomy 0.000 title claims abstract description 251
- 238000001514 detection method Methods 0.000 title claims abstract description 144
- 238000000034 method Methods 0.000 claims abstract description 72
- 238000000746 purification Methods 0.000 claims abstract description 59
- 238000005119 centrifugation Methods 0.000 claims abstract description 10
- 239000002245 particle Substances 0.000 claims description 67
- 239000012474 protein marker Substances 0.000 claims description 51
- 239000004005 microsphere Substances 0.000 claims description 33
- 238000011534 incubation Methods 0.000 claims description 31
- 102000004169 proteins and genes Human genes 0.000 claims description 26
- 108090000623 proteins and genes Proteins 0.000 claims description 26
- 239000000047 product Substances 0.000 claims description 24
- 210000004027 cell Anatomy 0.000 claims description 23
- 102100025222 CD63 antigen Human genes 0.000 claims description 20
- 101000934368 Homo sapiens CD63 antigen Proteins 0.000 claims description 20
- 238000000684 flow cytometry Methods 0.000 claims description 19
- 238000010586 diagram Methods 0.000 claims description 18
- 108010052285 Membrane Proteins Proteins 0.000 claims description 16
- 102000018697 Membrane Proteins Human genes 0.000 claims description 16
- 239000007850 fluorescent dye Substances 0.000 claims description 14
- 230000027455 binding Effects 0.000 claims description 13
- 238000003745 diagnosis Methods 0.000 claims description 12
- 239000002244 precipitate Substances 0.000 claims description 11
- 238000000695 excitation spectrum Methods 0.000 claims description 10
- 238000000295 emission spectrum Methods 0.000 claims description 8
- 239000011859 microparticle Substances 0.000 claims description 8
- 238000003556 assay Methods 0.000 claims description 7
- 239000002502 liposome Substances 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- 101710165845 CD81 protein Proteins 0.000 claims description 5
- 102100037904 CD9 antigen Human genes 0.000 claims description 5
- 201000010099 disease Diseases 0.000 claims description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 4
- 230000001225 therapeutic effect Effects 0.000 claims 1
- 238000000605 extraction Methods 0.000 abstract description 16
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 11
- 230000008569 process Effects 0.000 abstract description 4
- 239000000523 sample Substances 0.000 description 72
- 239000000243 solution Substances 0.000 description 23
- 230000000052 comparative effect Effects 0.000 description 19
- 239000012099 Alexa Fluor family Substances 0.000 description 18
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 17
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 17
- 201000002528 pancreatic cancer Diseases 0.000 description 17
- 208000008443 pancreatic carcinoma Diseases 0.000 description 17
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 15
- 101150030514 GPC1 gene Proteins 0.000 description 13
- 108010004729 Phycoerythrin Proteins 0.000 description 12
- 239000011324 bead Substances 0.000 description 12
- 239000012528 membrane Substances 0.000 description 12
- 238000005199 ultracentrifugation Methods 0.000 description 11
- 239000002202 Polyethylene glycol Substances 0.000 description 8
- 229920001223 polyethylene glycol Polymers 0.000 description 8
- 108010004469 allophycocyanin Proteins 0.000 description 6
- 238000010168 coupling process Methods 0.000 description 6
- 238000011002 quantification Methods 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- AFCARXCZXQIEQB-UHFFFAOYSA-N N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CCNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 AFCARXCZXQIEQB-UHFFFAOYSA-N 0.000 description 5
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 5
- 239000012535 impurity Substances 0.000 description 5
- 238000001556 precipitation Methods 0.000 description 5
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 5
- 239000012103 Alexa Fluor 488 Substances 0.000 description 4
- 239000012110 Alexa Fluor 594 Substances 0.000 description 4
- 239000012114 Alexa Fluor 647 Substances 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000003149 assay kit Methods 0.000 description 4
- 210000001124 body fluid Anatomy 0.000 description 4
- 239000010839 body fluid Substances 0.000 description 4
- 230000008878 coupling Effects 0.000 description 4
- 238000005859 coupling reaction Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000000635 electron micrograph Methods 0.000 description 4
- 239000011259 mixed solution Substances 0.000 description 4
- 238000004445 quantitative analysis Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000004062 sedimentation Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000004627 transmission electron microscopy Methods 0.000 description 4
- 238000000108 ultra-filtration Methods 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 3
- 230000005540 biological transmission Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 238000000432 density-gradient centrifugation Methods 0.000 description 3
- 150000002632 lipids Chemical class 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 238000001542 size-exclusion chromatography Methods 0.000 description 3
- HMUNWXXNJPVALC-UHFFFAOYSA-N 1-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)C(CN1CC2=C(CC1)NN=N2)=O HMUNWXXNJPVALC-UHFFFAOYSA-N 0.000 description 2
- YLZOPXRUQYQQID-UHFFFAOYSA-N 3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-1-[4-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]piperazin-1-yl]propan-1-one Chemical compound N1N=NC=2CN(CCC=21)CCC(=O)N1CCN(CC1)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F YLZOPXRUQYQQID-UHFFFAOYSA-N 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000031018 biological processes and functions Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 2
- 238000003759 clinical diagnosis Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000002284 excitation--emission spectrum Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000035992 intercellular communication Effects 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000012264 purified product Substances 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- LDXJRKWFNNFDSA-UHFFFAOYSA-N 2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-1-[4-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]piperazin-1-yl]ethanone Chemical compound C1CN(CC2=NNN=C21)CC(=O)N3CCN(CC3)C4=CN=C(N=C4)NCC5=CC(=CC=C5)OC(F)(F)F LDXJRKWFNNFDSA-UHFFFAOYSA-N 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 108010004103 Chylomicrons Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 102100026236 Interleukin-8 Human genes 0.000 description 1
- 108010007622 LDL Lipoproteins Proteins 0.000 description 1
- 102000007330 LDL Lipoproteins Human genes 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 241001122767 Theaceae Species 0.000 description 1
- 101710098580 Tubulin gamma-1 chain Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 210000003722 extracellular fluid Anatomy 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 229920001600 hydrophobic polymer Polymers 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 102000006240 membrane receptors Human genes 0.000 description 1
- 108020004084 membrane receptors Proteins 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000013610 patient sample Substances 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Pathology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Dispersion Chemistry (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to a flow detection method of exosomes and application thereof, belonging to the technical field of molecular biology. The invention provides a flow detection method of exosomes, which comprises the following purification steps: and centrifuging the sample to be detected for 10-20 min at 300-3000 g, and centrifuging for 15-30 min at 8000-18000 g to obtain a treated solution sample containing exosomes. The purification method utilizes two steps of centrifugation at different conventional speeds, so that the processed sample can be detected on machine, the dependence of the detection process on expensive large-scale instruments (such as an ultracentrifuge) and extraction reagents is greatly reduced, and the detection cost of exosomes is reduced; in addition, the purification method can enable the processed sample to be detected on the machine only by centrifugation at a conventional speed of 30 min, so that the detection time is greatly shortened, and the detection efficiency of exosomes is improved.
Description
Technical Field
The invention relates to a flow detection method of exosomes and application thereof, belonging to the technical field of molecular biology.
Background
Exosomes (exosomes) are a class of small, membrane-encapsulated, vesicular structures released by a variety of cells, which naturally occur in body fluids including blood, saliva, urine, cerebrospinal fluid and milk, but also can be found in extracellular environments such as cell culture media, interstitial fluid and the like. The exosomes generally have a size ranging from 30 to 300 nanometers, are composed of lipid bilayer of cell membranes, and contain cell components such as proteins, RNAs, DNAs, metabolites, and the like.
Research shows that exosomes can participate in various biological processes including intercellular communication, virus transmission, immune response and tumor occurrence through carrying, transmitting and interacting intracellular information molecules such as miRNAs, mRNAs, proteins and the like, and the exosomes are one of important ways of intercellular communication and carry a large amount of biological information. Meanwhile, the exosomes have a membrane coating structure so that the exosomes can stably exist in body fluid. Therefore, the exosomes can reflect the state of cells and the change of biological processes, are potential disease diagnosis markers, and have important research values in the fields of cell biology and molecular medicine.
At present, exosomes in a sample to be detected are extracted first, and then the exosomes can be detected. Common exosome extraction methods mainly comprise size exclusion chromatography, ultracentrifugation, density gradient centrifugation, ultrafiltration membrane separation, polymer sedimentation, immunoseparation, and the like. Among them, size exclusion chromatography is a chromatography technique for achieving separation based on the difference in retention of analytes of different molecular sizes in the pores of a chemically inert porous stationary phase, which is suitable for large-scale extraction of exosomes, has high separation purity, but requires a large amount of extraction reagents such as gel and organic solvent, and is costly, and some particles of similar size, such as chylomicron, low density lipoprotein, etc., are also purified.
The ultracentrifugation method is a method of separating, concentrating, and purifying a substance by applying a strong centrifugal force (generally, it is called ultracentrifugation at a rotational speed of 20000 and g or more) according to a difference in sedimentation coefficient, mass, density, or the like of the substance, and it is necessary to use an expensive ultracentrifuge and perform the ultracentrifugation for a long time (2 h).
The density gradient centrifugation method is to form a continuous or discontinuous density gradient in a centrifuge tube by using a certain medium, place cell suspension or homogenate on the top of the medium, and separate and delaminate cells by gravity or the action of a centrifugal force field, and the method can remove impurities such as large protein aggregates in a sample better by using the medium such as sucrose, so as to obtain purer exosomes, but has the defects of complicated operation, long time consumption (16-20 h) and unsuitable large-scale extraction.
The ultrafiltration membrane separation method filters a solvent and small molecular substances to the other side of the membrane, and retains the relative macromolecular substances on the ultrafiltration membrane so as to achieve the purpose of separation, and has the advantages of convenient operation, higher enrichment efficiency, easy collocation with other membranes or operation, lower cost, easy membrane filtration and easy deformation, membrane blockage, lower purity, serious pollution of large-particle protein, difficult recovery of exosomes attached to the membrane in the elution process, and extraction loss.
The polymer sedimentation method is characterized in that the solubility and the dispersibility of exosomes are changed through a high-molecular hydrophobic polymer such as polyethylene glycol (PEG), so that the exosomes can be settled out under a relatively low centrifugal force, the operation is simple, an ultracentrifuge is not needed, the obtained exosomes are more in quantity, and the method is also suitable for large-dose sample treatment, but the exosomes obtained by extraction of the exosomes have relatively high impurity content, and particularly, some hybrid proteins.
The immunoseparation method is a method for realizing specific enrichment of exosomes based on the specific binding action between the corresponding antibody and the marker protein, and has the advantages of higher exosome purity and smaller influence on exosome membrane structure, but the method requires a large amount of extraction reagents such as antibodies, magnetic beads and the like, has high cost, is not suitable for large-scale extraction, and has harsh storage conditions of the extraction reagents.
As can be seen, the usual exosome extraction methods such as size exclusion chromatography, ultracentrifugation, density gradient centrifugation, ultrafiltration membrane separation, polymer sedimentation, and immunoseparation generally involve expensive large-scale instruments and extraction reagents, and generally have problems of complicated operation and long time consumption, which are disadvantageous in terms of detection time, detection cost, and detection efficiency.
Common exotic detection methods mainly include Western Blot, flow cytometry, chemiluminescence, ELISA, protein mass spectrometry, colloidal gold, and chemical methods. Among them, flow CytoMetry (FCM) is a technique for performing high-speed, cell-by-cell quantitative analysis and sorting on single cells or other biological particles in suspension by detecting fluorescent signals of markers, and is characterized by quantitatively determining many important parameters such as cell DNA content, cell volume, protein content, enzyme activity, cell membrane receptors and surface antigens by rapidly determining coulter resistance, fluorescence, light scattering and light absorption. Compared with other detection methods, the method has the advantages that the size and the membrane protein of a single exosome can be characterized by using the flow cytometry to detect the exosome, the detection accuracy is higher, the detection duration is shorter, and the reagent adjustment is more convenient. However, in the existing exosome flow detection method, exosomes are coupled through magnetic beads, different numbers of exosomes can be coupled on each magnetic bead, quantitative analysis on specific proteins or lipids of a single exosome cannot be performed, and in addition, the sample preparation process comprises a plurality of incubation and washing steps, so that the loss of the exosomes is increased, meanwhile, the exosomes cannot be determined to be the exosomes by the magnetic bead coupling exosomes, and in addition, the complexity of the magnetic bead coupling process also increases the cost of exosome flow detection.
Disclosure of Invention
In order to solve the above problems, the present invention provides a flow detection method of exosomes, which is not for the purpose of diagnosis and treatment of diseases, comprising the steps of:
and (3) purification: centrifuging a sample to be detected for 10-20 min at 300-3000 g, and centrifuging for 15-30 min at 8000-18000 g to obtain a treated solution sample for detecting exosome flow type cells;
incubation: mixing a treated solution sample for detecting exosome flow type cells with a first flow type fluorescent antibody, and then incubating to obtain an incubation product; the first flow fluorescent antibody is capable of specifically binding to a first protein label; the first protein marker comprises surface proteins of exosomes;
the detection step comprises: performing flow detection on the incubated product by using a flow cytometer to obtain a flow detection result of the exosome;
alternatively, the streaming detection method includes the following steps:
and (3) purification: centrifuging a sample to be detected for 10-20 min at 300-3000 g, and centrifuging for 15-30 min at 8000-18000 g to obtain a treated solution sample for detecting exosome flow type cells;
incubation: mixing and incubating a treated solution sample for detecting exosome flow cytometry, a first flow fluorescent antibody and a second flow fluorescent antibody to obtain an incubation product; the first flow fluorescent antibody is capable of specifically binding to a first protein label; the first protein marker comprises surface proteins of exosomes; the second flow fluorescent antibody is capable of specifically binding to a second protein label, and the excitation spectrum and the emission spectrum of the fluorescent label on the second flow fluorescent antibody are different from the excitation spectrum and the emission spectrum of the fluorescent label on the first flow fluorescent antibody; the second protein marker comprises surface proteins and/or liposomes of exosomes;
The detection step comprises: and (3) performing flow detection on the incubated product by using a flow cytometer to obtain a flow detection result of the exosome.
In one embodiment of the invention, the purification step is: and centrifuging the sample to be detected for 10-20 min before 500-2000 g, and centrifuging for 15-30 min after 10000-15000 g to obtain a treated solution sample containing exosomes.
In one embodiment of the invention, the purification step is: the sample to be tested was centrifuged for 10 min at 2000 and g and for 20 min at 13500 and g to obtain a treated solution sample containing exosomes.
In one embodiment of the invention, the first protein marker comprises CD9 protein, CD81 protein and/or CD63 protein.
In one embodiment of the invention, the first flow fluorescent antibody comprises an Anti-CD9 flow fluorescent antibody, an Anti-CD81 flow fluorescent antibody and/or an Anti-CD63 flow fluorescent antibody.
In one embodiment of the invention, the first protein marker is CD63 protein.
In one embodiment of the invention, the first flow fluorescent antibody is an Anti-CD63 flow fluorescent antibody.
In one embodiment of the invention, the fluorescent label on the first flow fluorescent antibody comprises Fluorescein Isothiocyanate (FITC), alexa Fluor 488, alexa Fluor 594, alexa Fluor 647, cy5, phycoerythrin (PE), propidium Iodide (PI) and/or Allophycocyanin (APC).
In one embodiment of the invention, the second protein marker comprises GPC-1 protein.
In one embodiment of the invention, the second flow fluorescent antibody comprises an Anti-GPC1 flow fluorescent antibody.
In one embodiment of the invention, the fluorescent label on the second flow fluorescent antibody comprises fluorescein isothiocyanate, alexa Fluor 488, alexa Fluor 594, alexa Fluor 647, cy5, phycoerythrin, propidium iodide and/or allophycocyanin.
In one embodiment of the invention, in the incubating step, the incubating conditions are: incubating for 15-30 min at 15-25 ℃.
In one embodiment of the present invention, the detecting step is: firstly, adding calibration microspheres with diameters of 50-500 nanometers to a flow cytometer, distributing the calibration microspheres with different particle sizes at different positions by adjusting the gain and the threshold value of a detection channel, setting particles with the sizes of 50-500 nanometers to form a gate, adding an incubation product to the flow cytometer for flow detection, establishing a parameter scatter diagram of a first protein marker, finally setting a nano population for expressing the first protein marker, detecting vesicle microparticles (the exosomes in the gate positive for the first protein marker), and obtaining the particle number of the exosomes in a sample to be detected;
Alternatively, the detecting step includes: firstly, adding calibration microspheres with the diameter of 50-500 nanometers to a flow cytometer, distributing the calibration microspheres with different particle sizes at different positions by adjusting the gain and the threshold value of a detection channel, defining particles with the size of 50-500 nanometers, then adding an incubation product to the flow cytometer for flow detection, establishing a parameter scatter diagram of a first protein marker, defining a nano population for expressing the first protein marker, detecting vesicle microparticles contained in the door (the exosomes positive to the first protein marker), obtaining the particle number of the exosomes in a sample to be detected, finally establishing a parameter scatter diagram of a second protein marker from the exosomes population, defining a nano population for expressing the second protein marker, detecting vesicle microparticles contained in the door (the exosomes positive to the second protein marker), and obtaining the particle number of the exosomes positive to the second protein marker.
In one embodiment of the invention, the diameter of the calibration microsphere is 100-500 nanometers; the door is provided with: and (5) defining particles with the size of 100-500 nanometers and arranging a gate.
In one embodiment of the present invention, the sample to be tested is a body fluid.
In one embodiment of the invention, the body fluid comprises plasma, serum, urine, cerebrospinal fluid or alveolar lavage.
In one embodiment of the invention, the test sample is taken from an animal.
In one embodiment of the invention, the animal comprises a human, a mouse, a rabbit, a cow, a monkey, or a sheep.
The invention also provides a purification method of the exosome, which comprises the following steps: and centrifuging the sample to be detected for 10-20 min at 300-3000 g, and centrifuging for 15-30 min at 8000-18000 g to obtain a treated solution sample containing exosomes.
In one embodiment of the invention, the purification method comprises: and centrifuging the sample to be detected for 10-20 min before 500-2000 g, and centrifuging for 15-30 min after 10000-15000 g to obtain a treated solution sample containing exosomes.
In one embodiment of the invention, the purification method comprises: the sample to be tested was centrifuged for 10 min at 2000 and g and for 20 min at 13500 and g to obtain a treated solution sample containing exosomes.
The invention also provides a flow detection kit of the exosome, and the components of the flow detection kit comprise calibration microspheres and a first flow fluorescent antibody; the first flow fluorescent antibody is capable of specifically binding to a first protein label; the first protein marker comprises surface proteins of exosomes.
In one embodiment of the present invention, the diameter of the calibration microsphere is 50-500 nm.
In one embodiment of the present invention, the diameter of the calibration microsphere is 100 to 500 nanometers.
In one embodiment of the invention, the first protein marker comprises CD9 protein, CD81 protein and/or CD63 protein.
In one embodiment of the invention, the first flow fluorescent antibody comprises an Anti-CD9 flow fluorescent antibody, an Anti-CD81 flow fluorescent antibody and/or an Anti-CD63 flow fluorescent antibody.
In one embodiment of the invention, the first protein marker is CD63 protein.
In one embodiment of the invention, the first flow fluorescent antibody is an Anti-CD63 flow fluorescent antibody.
In one embodiment of the invention, the fluorescent label on the first flow fluorescent antibody comprises fluorescein isothiocyanate, alexa Fluor 488, alexa Fluor 594, alexa Fluor 647, cy5, phycoerythrin, propidium iodide and/or allophycocyanin.
In one embodiment of the invention, the components of the flow assay kit further comprise a second flow fluorescent antibody.
In one embodiment of the invention, the second flow fluorescent antibody is capable of specifically binding to a second protein label and the excitation spectrum and emission spectrum of the fluorescent label on the second flow fluorescent antibody are different from the excitation spectrum and emission spectrum of the fluorescent label on the first flow fluorescent antibody; the second protein marker comprises surface proteins and/or liposomes of the exosomes.
In one embodiment of the invention, the second protein marker comprises GPC-1 protein.
In one embodiment of the invention, the second flow fluorescent antibody comprises an Anti-GPC1 flow fluorescent antibody.
In one embodiment of the invention, the fluorescent label on the second flow fluorescent antibody comprises fluorescein isothiocyanate, alexa Fluor 488, alexa Fluor 594, alexa Fluor 647, cy5, phycoerythrin, propidium iodide and/or allophycocyanin.
The invention also provides a flow detection method of the exosome or a purification method of the exosome or application of a flow detection kit of the exosome in exosome detection, and the application is for diagnosis and treatment of non-diseases.
In one embodiment of the invention, the detection is a quantitative detection.
The technical scheme of the invention has the following advantages:
1. the invention provides a purification method of exosomes, which comprises the following steps: and centrifuging the sample to be detected for 10-20 min at 300-3000 g, and centrifuging for 15-30 min at 8000-18000 g to obtain a treated solution sample containing exosomes. The purification method utilizes two steps of centrifugation at different conventional speeds, so that the processed sample can be detected on machine, the dependence of the detection process on expensive large-scale instruments (such as an ultracentrifuge) and extraction reagents is greatly reduced, and the detection cost of exosomes is reduced; in addition, the purification method can enable the processed sample to be detected on the machine only by centrifugation at a conventional speed of 30 min, so that the detection time is greatly shortened, and the detection efficiency of exosomes is improved.
2. The invention provides a flow detection kit of exosomes, wherein the components of the flow detection kit comprise calibration microspheres and first flow fluorescent antibodies; the first flow fluorescent antibody is capable of specifically binding to a first protein label; the first protein marker comprises surface proteins of exosomes. The flow type detection kit can establish a flow type map with specific particle size through the specific-size calibration microspheres, and further, the detection range is defined in the specific particle size range (50-500 nm and the like), so that detection of a single exosome is facilitated, and the defect that different quantities of exosomes can be coupled on each magnetic bead by the existing exosome flow type detection method through coupling of the exosomes by the magnetic beads, and quantitative analysis of specific proteins or lipids of the single exosomes cannot be performed is overcome.
Further, the first protein marker is CD63 protein. The CD63 protein used in the flow detection kit can better represent almost all exosome subtypes, and researches show that the CD63 protein is used for marking particles with specific sizes, so that the particles with specific sizes can be further determined to be exosomes.
Further, the components of the flow assay kit further comprise a second flow fluorescent antibody; the second flow fluorescent antibody is capable of specifically binding to a second protein label, and the excitation spectrum and the emission spectrum of the fluorescent label on the second flow fluorescent antibody are different from the excitation spectrum and the emission spectrum of the fluorescent label on the first flow fluorescent antibody; the second protein marker comprises surface proteins and/or liposomes of the exosomes. The introduction of the second protein marker enables the flow detection kit to further capture information about surface proteins and/or liposomes on exosomes after positioning particles of different particle sizes based on a flow detection method.
Further, the second protein marker is GPC-1 protein. The introduction of GPC-1 protein enables the flow detection kit to further capture pancreatic cancer specific exosome protein information on exosomes after positioning particles with different particle diameters based on a flow detection method, so that noninvasive, rapid and convenient diagnosis of pancreatic cancer is realized, and meanwhile, the kit has the characteristics of high sensitivity and high specificity and can play a role in diagnosis of tumors in ultra-early or early stages.
3. The invention provides a flow detection method of exosomes, which comprises a purification step, an incubation step and a detection step; the purification steps are as follows: centrifuging a sample to be detected for 10-20 min at 300-3000 g, and centrifuging for 15-30 min at 8000-18000 g to obtain a treated solution sample for detecting exosome flow type cells; the incubation steps are as follows: mixing a treated solution sample for detecting exosome flow type cells with a first flow type fluorescent antibody, and then incubating to obtain an incubation product; the first flow fluorescent antibody is capable of specifically binding to a first protein label; the first protein marker comprises surface proteins of exosomes; the detection steps are as follows: and (3) performing flow detection on the incubated product by using a flow cytometer to obtain a flow detection result of the exosome. The purification step of the flow detection method utilizes two steps of centrifugation at different conventional speeds, so that the processed sample can be detected on the machine, the dependence of the detection process on expensive large-scale instruments (such as an ultracentrifuge) and extraction reagents is greatly reduced, and the detection cost of exosomes is reduced; in addition, the purification step of the flow detection method only needs conventional speed centrifugation for 30 min, so that the processed sample can be detected on the machine, the detection time is greatly shortened, and the detection efficiency of exosomes is improved; meanwhile, the flow detection method only needs three steps of purification, incubation and detection, and does not need to perform incubation and washing for a plurality of times as the existing exosome flow detection method, so that the loss of exosome is avoided; in addition, the flow detection method does not need to carry out magnetic bead coupling as the existing exosome flow detection method, avoids the complexity of a magnetic bead coupling process, and further reduces the cost of exosome flow detection.
Further, the detection steps are as follows: firstly, adding calibration microspheres with diameters of 50-500 nanometers to a flow cytometer, distributing the calibration microspheres with different particle sizes at different positions by adjusting the gain and the threshold value of a detection channel, setting particles with the sizes of 50-500 nanometers, adding an incubation product to the flow cytometer for flow detection, establishing a parameter scatter diagram of a first protein marker, finally setting a nano population for expressing the first protein marker, detecting vesicle microparticles contained in the gate (namely exosomes positive to the first protein marker in the gate), and obtaining the particle number of exosomes in a sample to be detected. According to the flow type detection method, a flow type map with specific particle size can be established through the specific-size calibration microspheres, so that the detection range is defined within the specific particle size range (50-500 nm), detection of a single exosome is facilitated, and the defect that the conventional exosome flow type detection method is capable of coupling exosome through magnetic beads, different numbers of exosome can be coupled on each magnetic bead, and quantitative analysis of specific proteins or lipids of the single exosome can not be performed is overcome.
Further, the first protein marker is CD63 protein. The CD63 protein used in the flow detection method can better represent almost all exosome subtypes, and researches show that the CD63 protein is used for marking particles with specific sizes, so that the particles with specific sizes can be further determined to be exosomes.
Further, the incubation step is as follows: mixing and incubating a treated solution sample for detecting exosome flow cytometry, a first flow fluorescent antibody and a second flow fluorescent antibody to obtain an incubation product; the first flow fluorescent antibody is capable of specifically binding to a first protein label; the first protein marker comprises surface proteins of exosomes; the second flow fluorescent antibody is capable of specifically binding to a second protein label, and the excitation spectrum and the emission spectrum of the fluorescent label on the second flow fluorescent antibody are different from the excitation spectrum and the emission spectrum of the fluorescent label on the first flow fluorescent antibody; the second protein marker comprises surface proteins and/or liposomes of the exosomes. The introduction of the second protein marker enables the flow detection method to further capture relevant information of surface proteins and/or liposomes on exosomes after positioning particles of different particle sizes.
Further, the second protein marker is GPC-1 protein. The introduction of GPC-1 protein enables the flow detection method to further capture pancreatic cancer specific exosome protein information on exosomes after positioning particles with different particle diameters, thereby realizing noninvasive, rapid and convenient diagnosis of pancreatic cancer, and simultaneously having the characteristics of high sensitivity and high specificity, and being capable of playing a role in diagnosis in the ultra-early or early stage of tumor.
Drawings
Fig. 1: electron microscopy results from different exosome purification methods were compared. In FIG. 1, A is an electron micrograph of an exosome purified by the purification method of comparative example 1-1 (ultracentrifugation), B is an electron micrograph of an exosome purified by the purification method of example 1-1, C is an electron micrograph of an exosome purified by the purification method of comparative example 1-2 (PEG precipitation), and D is an electron micrograph of an exosome purified by the purification method of comparative example 1-3.
Fig. 2: comparison of particle size distribution results for different exosome purification methods. In FIG. 2, A is the particle size distribution of the exosome purified by the purification method of comparative example 1-1 (ultracentrifugation), B is the particle size distribution of the exosome purified by the purification method of example 1-1, C is the particle size distribution of the exosome purified by the purification method of comparative example 1-2 (PEG precipitation), and D is the particle size distribution of the exosome purified by the purification method of comparative example 1-3.
Fig. 3: different sizes were used to demarcate the range of the microsphere-circumscribing exosomes.
Fig. 4: exosome flow assay results using CD9 protein as a marker.
Fig. 5: exosome flow assay using CD81 protein as a label.
Fig. 6: exosome flow assay results using CD63 protein as a marker.
Fig. 7: detection results of pancreatic cancer sample detection using CD63 and GPC-1 antibodies using flow cytometry.
Fig. 8: subject working curves for pancreatic cancer sample detection using CD63 and GPC-1 antibodies using flow cytometry.
Fig. 9: detection results of early pancreatic cancer sample detection using CD63 and GPC-1 antibodies using flow cytometry.
Fig. 10: subject working curves for early pancreatic cancer sample detection using CD63 and GCP-1 antibodies using flow cytometry.
Detailed Description
The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.
The following examples do not identify specific experimental procedures or conditions, which may be followed by procedures or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.
In the following examples, centrifugation at a speed below 20000 and g was performed by a conventional centrifuge (available from Semer, model Fresco 17) and centrifugation at a speed above 20000 and g was performed by an ultracentrifuge (available from Beckmann, model OPTIMA XPN-100); the exosome flow assays referred to in the examples below were performed by a beckmann DxFLEX series flow cytometer; the calibration microspheres referred to in the examples below are Megamix-Plus FSC beads available from Beckman Coulter, U.S.A.; the Anti-CD63 fluorescent antibodies, anti-CD9 fluorescent antibodies, and Anti-CD81 fluorescent antibodies referred to in the examples below are flow-type fluorescent antibodies available from St. Joule Biotechnology; GPC-1 antibodies referred to in the examples below were purchased from Abcam corporation.
Example 1-1: purification method of exosome
The embodiment provides a purification method of exosomes, which comprises the following steps: and centrifuging the sample to be detected for 10 min before 2000 and g, centrifuging for 20 min at 13500 and g, and discarding the precipitate to obtain the treated solution sample for exosome flow cell detection.
Examples 1-2: purification method of exosome
The embodiment provides a purification method of exosomes, which comprises the following steps: and (3) centrifuging the sample to be detected for 10 min at 300g, centrifuging for 20 min at 18000g, and discarding the precipitate to obtain a treated solution sample for exosome flow cell detection.
Examples 1-3: purification method of exosome
The embodiment provides a purification method of exosomes, which comprises the following steps: and (3) centrifuging the sample to be detected for 10 min before 3000 g, centrifuging for 20 min at 8000g, and discarding the precipitate to obtain a treated solution sample for exosome flow cell detection.
Examples 1 to 4: purification method of exosome
The embodiment provides a purification method of exosomes, which comprises the following steps: and centrifuging the sample to be detected for 10 min before 500 and g, centrifuging for 20 min after 15000 and g, and discarding the precipitate to obtain the treated solution sample for exosome flow cell detection.
Examples 1 to 5: purification method of exosome
The embodiment provides a purification method of exosomes, which comprises the following steps: and centrifuging the sample to be detected for 10 min before 2000 g, centrifuging for 20 min at 10000 g, and discarding the precipitate to obtain a treated solution sample for exosome flow cell detection.
Comparative examples 1-1: purification method of exosome (ultracentrifugation method)
The comparative example provides a purification method of exosomes, which is: the sample to be tested is centrifuged for 10 min (removing cells) at 300 g, then centrifuged for 10 min at 2000 g (removing dead cell fragments), then centrifuged for 10 min at 10000 g, finally centrifuged for 90 min at 120000 g, and the precipitate is taken out, thus obtaining the exosome sample which can be used for exosome flow cytometry detection.
Comparative examples 1-2: purification method of exosome (PEG precipitation method)
The comparative example provides a purification method of exosomes, which is: and centrifuging the sample to be detected for 10 min before 2000 g, adding an exosome extraction reagent (purchased from the Siemens Fedder company) in an adding amount which is 1/5 of the total volume of the sample to be detected, uniformly mixing, reacting at the temperature of 4 ℃ for 30 min, and taking a precipitate to obtain the exosome sample for exosome flow cytometry detection.
Comparative examples 1-3: purification method of exosome
The comparative example provides a purification method of exosomes, which is: and (3) centrifuging the sample to be detected for 10 min before 300 and g, centrifuging for 20 min at 2000 and g, and discarding the precipitate to obtain the treated solution sample for exosome flow cell detection.
Experimental example 1: influence of purification methods on the purification effect of exosomes
Taking 20 healthy human blood samples from a Nanjing drummer hospital as samples to be tested, purifying exosomes in the samples to be tested by using exosome purification methods of the embodiment 1-1 and the comparative embodiment 1-3 respectively, observing the purified products by using a transmission electron microscope, and carrying out Nanosight particle size tracking analysis on the purified products by using an NS300 nanoparticle tracking analyzer, wherein the observation results are shown in fig. 1-2.
As can be seen from fig. 1 to 2: transmission electron microscopy observation shows that similar to the exosomes obtained by the ultracentrifugation method of comparative example 1-1, the exosomes extracted in this example 1-1 show a tea-tray-like double-layer membrane structure under the transmission electron microscopy, and Nanosight particle size tracking analysis shows that the exosomes purified by the ultracentrifugation methods of example 1-1 and comparative example 1-1 and the exosomes obtained in this example have particle sizes of 50-200 nm, and the particle number in each milliliter reaches 10 7 However, the ultracentrifugation method of comparative example 1-1 takes 120 min, which is much higher than 30 min of example 1-1; transmission electron microscopy observation shows that the exosomes extracted in example 1-1 show a tea tray-like double-layer membrane structure under a transmission electron microscope similar to the exosomes obtained by the PEG precipitation method in comparative example 1-2, but the PEG precipitation method in comparative example 1-2 has impurity interference due to the influence of a PEG precipitant, and Nanosight particle size tracking analysis shows that the extracted exosomes in comparative example 1-2 have larger particle size; transmission electron microscopy shows that compared with the exosomes obtained by the method of the embodiment 1-1, the exosomes extracted in the embodiment 1-3 have a large amount of impurity interference, and the particle size of the particles is larger, and Nanosight particle size tracking analysis shows that the particle size of the exosomes extracted in the embodiment 1-3 is 100-400 nm, which indicates that the exosomes extracted in the mode have a large amount of impurity interference.
Example 2-1: flow type detection kit for exosome (exosome quantification)
The embodiment provides a flow detection kit for exosomes, which consists of nanometer calibration microspheres with different particle sizes and a first flow fluorescent antibody; the diameter of the calibrated microsphere is 100-500 nanometers (100 nanometers/200 nanometers/300 nanometers/500 nanometers), and the first flow type fluorescent antibody is an Anti-CD63 fluorescent antibody marked by Alexa Fluor ™ 594.
Example 2-2: flow type detection kit for exosome (exosome quantification)
The present example provides a flow assay kit for exosomes, which replaces the first flow fluorescent antibody with an Anti-CD63 fluorescent antibody labeled by Alexa Fluor ™ 594 with an Anti-CD9 fluorescent antibody labeled by Alexa Fluor ™ 594, based on example 2-1.
Examples 2-3: flow type detection kit for exosome (exosome quantification)
The present example provides a flow assay kit for exosomes, which replaces the first flow fluorescent antibody with an Anti-CD63 fluorescent antibody labeled by Alexa Fluor ™ 594 with an Anti-CD81 fluorescent antibody labeled by Alexa Fluor ™ 594, based on example 2-1.
Example 3-1: flow detection method of exosome (exosome quantification)
The embodiment provides a method for detecting exosomes in a flow mode, the method for detecting exosomes in a flow mode firstly extracts exosomes from a sample to be detected by using the purification method of the embodiment 1-1, and then detects exosomes extracted from the sample to be detected in a flow mode by using the flow detection kit of the embodiment 2-1, and the method comprises the following steps:
Step one: extracting exosomes from the sample to be tested using the purification method of example 1-1, obtaining a treated solution sample useful for exosome flow cytometry;
step two: adding 2 mu L of Alexa Fluor ™ 594 labeled Anti-CD63 fluorescent antibody into 100 mu L of treated solution sample for detecting exosome flow cytometry, and incubating at 25 ℃ for 30 min to obtain an incubation product;
step three: performing flow detection on the incubated product by using a flow cytometer to obtain a flow detection result of the exosome; when the flow cytometry is used for detecting the incubation products in a flow mode, standard calibration microspheres (100 nanometers/200 nanometers/300 nanometers/500 nanometers) with different particle sizes are firstly mixed with PBS buffer (concentration 0.01M, pH 7.4) according to the volume ratio of 1:10000 Diluting (standard calibration microsphere: PBS buffer solution) in proportion to prepare a calibration microsphere diluted mixed solution, adding 500 mu L of the calibration microsphere diluted mixed solution into a flow cytometer, regulating the gain and threshold of a detection channel to enable calibration microspheres with different particle sizes to be distributed at different positions, defining particles with the size of 100-500 nanometers (shown in figure 3), adding 100 mu L of an incubation product into the flow cytometer for flow detection, establishing a FSC/VSSC scatter diagram (FSC/VSSC scatter diagram) of a first protein marker, wherein an x-axis represents a VSSC value, a y-axis represents a FSC value, and represents the particle size distribution of an exosome) and a PE/VSSC scatter diagram (PE/VSSC scatter diagram, wherein an x-axis represents a VSSC value, and represents the content of the exosome), finally defining a nano population for expressing CD63 protein, and detecting vesicle microparticles contained in the door (the exosome positive to-be-tested CD63 protein) to obtain the number of exosome in a sample.
Example 3-2: flow detection method for exosomes
The present example provides a method for detecting exosomes in a flow format, which uses the flow detection kit of example 2-2 on the basis of example 3-1, replaces the Anti-CD63 fluorescent antibody labeled with Alexa Fluor ™ 594 with the Anti-CD9 fluorescent antibody labeled with Alexa Fluor ™ 594, and circles a nano population expressing CD9 protein to obtain the number of exosomes particles in the sample to be detected.
Examples 3-3: flow detection method for exosomes
The present embodiment provides a method for detecting exosomes in a flow format, which uses the flow detection kit of embodiment 2-3 on the basis of embodiment 3-1, replaces the Anti-CD63 fluorescent antibody labeled with Alexa Fluor ™ 594 with the Anti-CD81 fluorescent antibody labeled with Alexa Fluor ™ 594, and circles a nano population expressing CD81 protein to obtain the particle number of exosomes in the sample to be detected.
Experimental example 2: influence of protein marker selection on exosome flow detection effect
Taking 20 healthy human blood samples from a Nanjing drummer hospital as samples to be detected, and respectively carrying out flow detection on exosomes in the samples to be detected by using the exosome flow detection methods of the embodiments 3-1 to 3-3, wherein the detection results are shown in figures 4 to 6.
As can be seen from fig. 4 to 6: the flow detection of exosomes in the sample to be detected can be realized by using the flow detection kit of examples 2-1 to 2-3, wherein when the exosomes extracted in example 1-1 are detected by using the Anti-CD63 fluorescent antibody marked by Alexa Fluor ™ 594 (fig. 4), the content of the detected exosomes is up to 68.44% (fig. 4), and when the exosomes of the same sample extracted by the same extraction method are captured by using the Anti-9 fluorescent antibody marked by Alexa Fluor ™ 594, the capturing efficiency is 10.76% (fig. 5), only accounting for 1/7 of the Anti-CD63, and when the exosomes extracted by using the Anti-81 fluorescent antibody marked by Alexa Fluor ™ 594 are captured, the positive exosomes amount accounts for 6.65% (fig. 6), the capturing efficiency is about 1/10 of the Anti-CD63, and the exosomes marked by using Alexa Fluor ™ can be obtained, thereby being beneficial to the downstream application of the exosomes.
Examples 2 to 4: flow detection kit for exosomes (exosome quantification + pancreatic cancer diagnosis)
The embodiment provides a flow detection kit for exosomes, which consists of nanometer calibration microspheres with different particle sizes, a first flow fluorescent antibody and a second flow fluorescent antibody; the diameter of the calibrated microsphere is 100-500 nanometers (100 nanometers/200 nanometers/300 nanometers/500 nanometers), the first flow type fluorescent antibody is an Anti-CD63 fluorescent antibody marked by Alexa Fluor ™ 594, and the second flow type fluorescent antibody is an Anti-GPC1 marked fluorescent antibody of Alexa Fluor ™ 647.
Examples 3-4: flow detection method of exosome (exosome quantification + pancreatic cancer diagnosis)
The embodiment provides a method for detecting exosomes in a flow mode, the method firstly uses the purification method of the embodiment 1-1 to extract exosomes from a sample to be detected, and then uses the flow detection kit of the embodiment 2-4 to detect exosomes extracted from the sample to be detected in a flow mode, and the method comprises the following steps:
step one: extracting exosomes from the sample to be tested using the purification method of example 1-1, obtaining a treated solution sample useful for exosome flow cytometry;
step two: adding 2 mu L of Alexa Fluor ™ 594 labeled Anti-CD63 fluorescent antibody and 2 mu L of Alexa Fluor ™ 647 Anti-GPC1 labeled fluorescent antibody into 100 mu L of treated solution sample for exosome flow cytometry detection, and incubating at 25 ℃ for 30 min to obtain an incubation product;
step three: performing flow detection on the incubated product by using a flow cytometer to obtain a flow detection result of the exosome; when the flow cytometry is used for detecting the incubation products in a flow mode, standard calibration microspheres (100 nanometers/200 nanometers/300 nanometers/500 nanometers) with different particle sizes are firstly mixed with PBS buffer (concentration 0.01M, pH 7.4) according to the volume ratio of 1:10000 Diluting (standard calibration microsphere: PBS buffer solution) in proportion to prepare a calibration microsphere diluted mixed solution, adding 500 mu L of the calibration microsphere diluted mixed solution into a flow cytometer, regulating the gain and the threshold value of a detection channel to enable calibration microspheres with different particle sizes to be distributed at different positions, defining particles with the size of 100-500 nanometers (shown in figure 3), adding 100 mu L of an incubation product into the flow cytometer for flow detection, establishing a FSC/VSSC scatter diagram (FSC/VSSC scatter diagram) of a first protein marker, wherein an x-axis represents a VSSC value, a y-axis represents a VSSC value, represents particle size distribution of an exosome) and a PE/VSSC scatter diagram (PE/VSSC scatter diagram, wherein the x-axis represents a VSSC value, the y-axis represents a PE value, represents content of exosome), defining a nano population for expressing CD63 protein, wherein the micro-bubble particles contained in the detection gate (namely the exosome positive for CD63 protein) are arranged in different positions, obtaining the number of exosome in a sample, finally establishing a FSC/VSSC scatter diagram (FSC/VSSC scatter diagram) of a first protein marker, and finally establishing a positive ligand (APC/PE 1) from the figure in the GPC scatter diagram represents the nano-axis represents the exosome positive particle of the protein marker, wherein the positive ligand of the micro-protein marker is expressed in the GPC (PE 1).
Experimental example 3: verification of pancreatic cancer diagnostic Effect
The exosomes in the samples to be tested were subjected to flow detection by using the exosome flow detection method of examples 3-4 with 50 clinical diagnosis of pancreatic cancer patients of stage i-iv and 50 clinical diagnosis of healthy persons (control) corresponding to the gender and age of pancreatic cancer patients as the samples to be tested, and the detection results are shown in fig. 7-10.
As can be seen from fig. 7 to 10, the healthy control samples have 0 to 20 GPC 1-positive exosomes, which is an average of 11 GPC 1-positive exosomes, whereas the pancreatic cancer patient samples exhibit a higher proportion of GPC 1-positive exosomes (fig. 7, p < 0.0001), suggesting that the flow detection kit of examples 2 to 4 and the exosome flow detection method of examples 3 to 4 can be applied to diagnosis and screening of pancreatic cancer, the ROC curve results show that the sensitivity for pancreatic cancer diagnosis is 90%, the specificity is 86%, the area under the curve is as high as 0.9404 (fig. 8), the sensitivity for early diagnosis of pancreatic cancer is 84%, and the specificity is: 80%, area under curve: 0.8645 (FIGS. 9 to 10).
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (5)
1. A flow assay for exosomes, wherein the flow assay is not diagnostic or therapeutic for disease, the flow assay comprising the steps of:
and (3) purification: centrifuging at two different conventional speeds for 10-20 min, centrifuging at 10000-15000 g for 15-30 min, and discarding the precipitate to obtain a treated solution sample for exosome flow cell detection;
incubation: mixing a treated solution sample for detecting exosome flow type cells with a first flow type fluorescent antibody, and then incubating to obtain an incubation product; the first flow fluorescent antibody is capable of specifically binding to a first protein label; the first protein marker comprises surface proteins of exosomes;
the detection step comprises: firstly, adding calibration microspheres with diameters of 50-500 nanometers to a flow cytometer, distributing the calibration microspheres with different particle sizes at different positions by adjusting the gain and the threshold value of a detection channel, setting particles with the sizes of 50-500 nanometers to form a gate, adding an incubation product to the flow cytometer for flow detection, establishing a parameter scatter diagram of a first protein marker, finally setting a nano population for expressing the first protein marker, detecting vesicle microparticles contained in the gate, and obtaining the particle number of exosomes in a sample to be detected;
The flow detection method only needs three steps of purification, incubation and detection, wherein the purification step only needs to utilize two steps of centrifugation at different conventional speeds, and multiple times of incubation and washing are not needed;
alternatively, the streaming detection method includes the following steps:
and (3) purification: centrifuging at two different conventional speeds for 10-20 min, centrifuging at 10000-15000 g for 15-30 min, and discarding the precipitate to obtain a treated solution sample for exosome flow cell detection;
incubation: mixing and incubating a treated solution sample for detecting exosome flow cytometry, a first flow fluorescent antibody and a second flow fluorescent antibody to obtain an incubation product; the first flow fluorescent antibody is capable of specifically binding to a first protein label; the first protein marker comprises surface proteins of exosomes; the second flow fluorescent antibody is capable of specifically binding to a second protein label, and the excitation spectrum and the emission spectrum of the fluorescent label on the second flow fluorescent antibody are different from the excitation spectrum and the emission spectrum of the fluorescent label on the first flow fluorescent antibody; the second protein marker comprises surface proteins and/or liposomes of exosomes;
The detection step comprises: firstly, adding calibration microspheres with diameters of 50-500 nanometers to a flow cytometer, distributing the calibration microspheres with different particle sizes at different positions by adjusting the gain and the threshold value of a detection channel, defining particles with the sizes of 50-500 nanometers, arranging a gate, adding an incubation product to the flow cytometer for flow detection, establishing a parameter scatter diagram of a first protein marker, defining a nano population for expressing the first protein marker, detecting vesicle microparticles contained in the gate to obtain the number of particles of an exosome in a sample to be detected, finally establishing a parameter scatter diagram of a second protein marker from the exosome population, defining a nano population for expressing the second protein marker, detecting the vesicle microparticles contained in the gate to obtain the number of particles of the exosome positive to the second protein marker;
the flow detection method only needs three steps of purification, incubation and detection, wherein the purification step only needs to utilize two steps of centrifugation at different conventional speeds, and multiple incubation and washing are not needed.
2. The method of claim 1, wherein the first protein label comprises CD9 protein, CD81 protein and/or CD63 protein; the first flow fluorescent antibody comprises an Anti-CD9 flow fluorescent antibody, an Anti-CD81 flow fluorescent antibody and/or an Anti-CD63 flow fluorescent antibody.
3. The method for detecting exosomes according to claim 1 or 2, wherein the diameter of the calibration microspheres is 100-500 nm; the door is provided with: and (5) defining particles with the size of 100-500 nanometers and arranging a gate.
4. The method for the flow-through detection of exosomes according to claim 1 or 2, wherein the purification step comprises: the sample to be tested is centrifuged for 10 min before 2000 and g, and is centrifuged for 20 min at 13500 and g, and the precipitate is discarded, thus obtaining the treated solution sample containing exosomes.
5. The use of the flow detection method of exosomes according to any one of claims 1-4 in exosome detection, characterized in that the use is for non-disease diagnosis and treatment purposes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311596892.1A CN117330481B (en) | 2023-11-27 | 2023-11-27 | Flow detection method for exosomes and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311596892.1A CN117330481B (en) | 2023-11-27 | 2023-11-27 | Flow detection method for exosomes and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117330481A CN117330481A (en) | 2024-01-02 |
| CN117330481B true CN117330481B (en) | 2024-04-09 |
Family
ID=89283341
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311596892.1A Active CN117330481B (en) | 2023-11-27 | 2023-11-27 | Flow detection method for exosomes and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117330481B (en) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106290877A (en) * | 2016-09-08 | 2017-01-04 | 北京海思特临床检验所有限公司 | Acute myeloid leukemia G-Ag expression detection kit and detection method |
| CN108885210A (en) * | 2016-02-05 | 2018-11-23 | 纳诺韦尔诊断有限公司 | The detection of allochthon with surface marker |
| CN109324186A (en) * | 2018-11-08 | 2019-02-12 | 上海宝藤生物医药科技股份有限公司 | Excretion body protein CD82, GPC1 combine CA19-9 for cancer of pancreas early diagnosis and curative effect monitoring |
| CN109991427A (en) * | 2019-04-08 | 2019-07-09 | 王延博 | It is a kind of for detecting the kit of excretion body surface face protein marker in serum |
| CN110702589A (en) * | 2019-10-11 | 2020-01-17 | 南京吉鹏博生物科技有限公司 | Method for detecting exosome by flow cytometer |
| CN112379096A (en) * | 2021-01-13 | 2021-02-19 | 江苏申基生物科技有限公司 | Application of exosome membrane protein as colon cancer diagnosis marker and colon cancer early diagnosis kit |
| CN112501173A (en) * | 2020-11-09 | 2021-03-16 | 苏州吉玛基因股份有限公司 | GPC1DNA aptamer and application thereof |
| CN115047182A (en) * | 2017-10-05 | 2022-09-13 | 香港科技大学 | Exosome analysis and cancer diagnosis method |
| CN117070452A (en) * | 2023-08-24 | 2023-11-17 | 吉林省肿瘤医院 | High-quality exosome extraction method for liquid biopsy of CD47 and DLL3 genes |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030008410A1 (en) * | 1995-03-13 | 2003-01-09 | Hechinger Mark K. | Immunoassay apparatus, kit and methods |
-
2023
- 2023-11-27 CN CN202311596892.1A patent/CN117330481B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108885210A (en) * | 2016-02-05 | 2018-11-23 | 纳诺韦尔诊断有限公司 | The detection of allochthon with surface marker |
| CN106290877A (en) * | 2016-09-08 | 2017-01-04 | 北京海思特临床检验所有限公司 | Acute myeloid leukemia G-Ag expression detection kit and detection method |
| CN115047182A (en) * | 2017-10-05 | 2022-09-13 | 香港科技大学 | Exosome analysis and cancer diagnosis method |
| CN109324186A (en) * | 2018-11-08 | 2019-02-12 | 上海宝藤生物医药科技股份有限公司 | Excretion body protein CD82, GPC1 combine CA19-9 for cancer of pancreas early diagnosis and curative effect monitoring |
| CN109991427A (en) * | 2019-04-08 | 2019-07-09 | 王延博 | It is a kind of for detecting the kit of excretion body surface face protein marker in serum |
| CN110702589A (en) * | 2019-10-11 | 2020-01-17 | 南京吉鹏博生物科技有限公司 | Method for detecting exosome by flow cytometer |
| CN112501173A (en) * | 2020-11-09 | 2021-03-16 | 苏州吉玛基因股份有限公司 | GPC1DNA aptamer and application thereof |
| CN112379096A (en) * | 2021-01-13 | 2021-02-19 | 江苏申基生物科技有限公司 | Application of exosome membrane protein as colon cancer diagnosis marker and colon cancer early diagnosis kit |
| CN117070452A (en) * | 2023-08-24 | 2023-11-17 | 吉林省肿瘤医院 | High-quality exosome extraction method for liquid biopsy of CD47 and DLL3 genes |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117330481A (en) | 2024-01-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhao et al. | Isolation and analysis methods of extracellular vesicles (EVs) | |
| Mohammadi et al. | Emerging technologies and commercial products in exosome-based cancer diagnosis and prognosis | |
| US11899024B2 (en) | Treatment and diagnosis of parkinson's disease using isolated and enriched populations of biofluid-derived extracellular vesicles | |
| Zhang et al. | Immunomagnetic sequential ultrafiltration (iSUF) platform for enrichment and purification of extracellular vesicles from biofluids | |
| US11833504B2 (en) | Microfluidic label-free isolation and identification of cells using fluorescence lifetime imaging (FLIM) | |
| JP6661368B2 (en) | Multi-sort cell separation method | |
| Zhang et al. | Characterization and applications of extracellular vesicle proteome with post-translational modifications | |
| CN110036111A (en) | The probe based on lipid for extracellularly separating | |
| CN111670067B (en) | Method for separating extracellular vesicles from biological material | |
| CN110702589A (en) | Method for detecting exosome by flow cytometer | |
| WO2019132688A1 (en) | Method and device for separating extracellular vesicles from biological liquids with the aid of cascade ultrafiltration | |
| CN111073846A (en) | Method for separating extracellular vesicles from tissue specific sources and kit thereof | |
| CN113774008A (en) | Method for extracting exosome and application thereof | |
| Pallares-Rusiñol et al. | Advances in exosome analysis | |
| JP7376021B2 (en) | Method for separating and analyzing microvesicles from human blood | |
| JP7444386B2 (en) | Method for isolating and analyzing microvesicles from human urine | |
| Wang et al. | A light-activated magnetic bead strategy utilized in spatio-temporal controllable exosomes isolation | |
| Lv et al. | Efficient detection of single circulating tumor cell in blood using Raman mapping based on Aptamer-SERS bio-probe coupled with micropore membrane filtration | |
| Malhotra et al. | Novel devices for isolation and detection of bacterial and mammalian extracellular vesicles | |
| CN117330481B (en) | Flow detection method for exosomes and application thereof | |
| US20140309553A1 (en) | Kits and methods for separating a target analyte from a suspension | |
| CN111575239A (en) | Method and device for enriching circulating tumor cells | |
| CN212560306U (en) | Enrichment device of circulating tumor cells | |
| CN110218693B (en) | Reagent combination, kit and method for extracting exosomes | |
| Noreldin et al. | Isolation and Characterization of Extracellular Vesicles: Classical and Modern Approaches |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |