CN111896751B - 一种高灵敏度目视检测外泌体的方法 - Google Patents
一种高灵敏度目视检测外泌体的方法 Download PDFInfo
- Publication number
- CN111896751B CN111896751B CN202010797874.XA CN202010797874A CN111896751B CN 111896751 B CN111896751 B CN 111896751B CN 202010797874 A CN202010797874 A CN 202010797874A CN 111896751 B CN111896751 B CN 111896751B
- Authority
- CN
- China
- Prior art keywords
- solution
- exosome
- filter membrane
- aptamer
- cus
- 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 68
- 238000000034 method Methods 0.000 title claims abstract description 15
- 230000035945 sensitivity Effects 0.000 title claims abstract description 9
- 239000000243 solution Substances 0.000 claims abstract description 80
- 239000012528 membrane Substances 0.000 claims abstract description 46
- 108091023037 Aptamer Proteins 0.000 claims abstract description 28
- 238000001514 detection method Methods 0.000 claims abstract description 28
- 238000006243 chemical reaction Methods 0.000 claims abstract description 20
- 230000000007 visual effect Effects 0.000 claims abstract description 16
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 14
- 230000008859 change Effects 0.000 claims abstract description 11
- ILWRPSCZWQJDMK-UHFFFAOYSA-N triethylazanium;chloride Chemical compound Cl.CCN(CC)CC ILWRPSCZWQJDMK-UHFFFAOYSA-N 0.000 claims abstract description 9
- 108010052285 Membrane Proteins Proteins 0.000 claims abstract description 7
- 239000004094 surface-active agent Substances 0.000 claims abstract description 7
- 102000018697 Membrane Proteins Human genes 0.000 claims abstract description 6
- 239000011259 mixed solution Substances 0.000 claims abstract description 3
- 239000002105 nanoparticle Substances 0.000 claims description 21
- 102100025222 CD63 antigen Human genes 0.000 claims description 14
- 101000934368 Homo sapiens CD63 antigen Proteins 0.000 claims description 14
- 101710134784 Agnoprotein Proteins 0.000 claims description 10
- 125000003396 thiol group Chemical group [H]S* 0.000 claims description 10
- 239000003446 ligand Substances 0.000 claims description 9
- 230000035484 reaction time Effects 0.000 claims description 7
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 3
- 102100027221 CD81 antigen Human genes 0.000 claims description 2
- 101000914479 Homo sapiens CD81 antigen Proteins 0.000 claims description 2
- 101001133056 Homo sapiens Mucin-1 Proteins 0.000 claims description 2
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 claims description 2
- 102100034256 Mucin-1 Human genes 0.000 claims description 2
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 claims description 2
- 238000011010 flushing procedure Methods 0.000 claims description 2
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 2
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 2
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 102000018651 Epithelial Cell Adhesion Molecule Human genes 0.000 claims 1
- 108010066687 Epithelial Cell Adhesion Molecule Proteins 0.000 claims 1
- 238000005406 washing Methods 0.000 abstract description 8
- 201000010099 disease Diseases 0.000 abstract description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 5
- 238000003745 diagnosis Methods 0.000 abstract description 4
- 206010028980 Neoplasm Diseases 0.000 abstract description 3
- 201000011510 cancer Diseases 0.000 abstract description 3
- 239000002245 particle Substances 0.000 abstract description 2
- 230000004044 response Effects 0.000 abstract description 2
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 abstract 2
- 230000002776 aggregation Effects 0.000 abstract 1
- 238000004220 aggregation Methods 0.000 abstract 1
- BWFPGXWASODCHM-UHFFFAOYSA-N copper monosulfide Chemical class [Cu]=S BWFPGXWASODCHM-UHFFFAOYSA-N 0.000 abstract 1
- OMZSGWSJDCOLKM-UHFFFAOYSA-N copper(II) sulfide Chemical compound [S-2].[Cu+2] OMZSGWSJDCOLKM-UHFFFAOYSA-N 0.000 abstract 1
- 239000000376 reactant Substances 0.000 abstract 1
- 229910001961 silver nitrate Inorganic materials 0.000 abstract 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 230000005540 biological transmission Effects 0.000 description 3
- 238000004737 colorimetric analysis Methods 0.000 description 3
- 239000007850 fluorescent dye Substances 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000035790 physiological processes and functions Effects 0.000 description 2
- 101150084967 EPCAM gene Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000008568 cell cell communication Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000000536 complexating effect Effects 0.000 description 1
- 230000008094 contradictory effect Effects 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
-
- 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
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
-
- 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
- G01N15/06—Investigating concentration of particle suspensions
- G01N15/0606—Investigating concentration of particle suspensions by collecting particles on a support
- G01N15/0618—Investigating concentration of particle suspensions by collecting particles on a support of the filter type
- G01N15/0625—Optical scan of the deposits
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- 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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- 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
- G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
-
- 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
- G01N2015/0038—Investigating nanoparticles
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Nanotechnology (AREA)
- Dispersion Chemistry (AREA)
- Plasma & Fusion (AREA)
- Zoology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Endocrinology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
本发明公开了一种高灵敏度目视检测外泌体的方法，通过将一定浓度的外泌体溶液与外泌体表面膜蛋白相应的适配体修饰的硫化铜反应一段时间，而后加入表面活性剂防止反应物粒子团聚，将反应溶液过50nm的滤膜留下外泌体和硫化铜连接体，用PBS洗涤滤膜、取出并加入硝酸银反应一段时间，最后加入三乙胺盐酸盐、3,3'，5,5'‑四甲基联苯胺以及过氧化氢混合溶液显色，经一定时间后观察滤膜的颜色变化。相对现有技术，本发明技术方案具有简单便捷且反应迅速等优点，可实现外泌体的高灵敏度目视检测。与此同时本发明技术方案的检测方法具有成本低廉、反应效率高以及稳定性极好等优点，能够在外泌体检测和疾病诊断，特别是癌症的检测中得到广泛应用。
Description
技术领域
本发明涉及分析化学和疾病诊断技术领域，特别涉及一种高灵敏度目视检测外泌体的方法。
背景技术
外泌体是细胞分泌的纳米级囊性小泡，在实现细胞与细胞之间通信交流起着重要作用。在正常的生理和病例状态，几乎所有的细胞都能够分泌外泌体，而发生癌变的细胞分泌的外泌体数量通常比正常的细胞分泌的外泌体高出几个数量级。
外泌体大小范围从约30nm至150nm，其表面含有多种膜蛋白，并且内部含有核酸，活性酶和细胞质底物。此外外泌体还包含许多蛋白质，这些蛋白质反映了细胞的表型和生理状态，且异质性很高。外泌体的上述特征可告知许多疾病的相关生理状态和病理过程，特别是癌症，因此对细胞分泌的外泌体的敏感识别对生物学研究和临床疾病诊断具有重要意义。
由于外泌体大小范围从约30nm至150nm，在普通的光学显微镜下无法检测，现有技术通常采用荧光染色方法或者流式细胞术来分析检测，然而复杂的荧光染色以及外泌体的弱光散射，从而限制这两种方法在外泌体检测中的应用。因此针对上述存在的技术问题，现有技术对于纳米级别的外泌体，还采用透射电子显微镜可视化分析和纳米颗粒跟踪分析，然而透射电子显微镜和纳米颗粒跟踪分析仪器价格昂贵，每次测试外泌体样品的价格大约为500元，而且透射电镜测量外泌体之前，还需要对外泌体进行染色，而纳米颗粒跟踪分析方法需要复杂的分离和纯化过程。
发明内容
本发明目的在于克服现有技术对外泌体检测的复杂前处理过程、昂贵的检测设备和检测费用以及无法区分干扰颗粒如蛋白质团聚体的技术缺陷，提出一种简单便捷且反应迅速的通过标记蛋白和外泌体特异性结合的快速检测外泌体方法，旨在实现外泌体快速且高灵敏度的目视检测。
为实现上述目的，本发明提出的一种高灵敏度目视检测外泌体的方法，包括以下步骤：
步骤S1：将CuS纳米粒子和带有巯基的外泌体表面膜蛋白适配体反应后得到CuS-膜蛋白适配体结合物；
步骤S2：将含有外泌体的溶液滤过200nm的滤膜，并向外泌体溶液中加入CuS-膜蛋白适配体结合物反应后得到外泌体-CuS结合物；
步骤S3：将步骤S2得到的反应液加入表面活性剂并通过50nm的滤膜，使用PBS冲洗滤膜得到含有外泌体-CuS结合物的滤膜；
步骤S4：向步骤S3得到的含有外泌体-CuS结合物的滤膜加入一定量的AgNO3溶液，反应后加入三乙胺盐酸盐、过氧化氢和3,3'，5,5'-四甲基联苯胺混合溶液反应显色，通过目视法观察滤膜的颜色变化。
优选地，所述步骤S1的CuS纳米粒子的大小为10～40nm，所述带有巯基的外泌体表面膜蛋白适配体为CD63适配体、CD81适配体、CD9适配体、EpCAM适配体、HER2适配体、MUC1适配体中的一种或多种；所述CuS纳米粒子和带有巯基的外泌体表面膜蛋白适配体的反应时间为10～24h。
优选地，所述步骤S2的外泌体溶液和CuS-膜蛋白适配体结合物反应时间为0.5～10h。
优选地，所述步骤S2的所述外泌体溶液通常为1mL，在外泌体溶液浓度通过提高外泌体溶液体积而提高灵敏度。
优选地，所述步骤S3的所述表面活性剂为十二烷基硫酸钠、十六烷基三甲基溴化铵、聚乙烯吡咯烷酮中的一种或多种，表面活性剂的浓度范围为0.1～2.0％。
优选地，所述步骤S4的所述AgNO3溶液浓度范围为10-5～10-3M，体积范围为10～50uL，步骤S4的所述的滤膜中物质和AgNO3反应时间范围为5～10min，所述三乙胺盐酸盐浓度为0.05～0.2M、过氧化氢浓度范围为0.1～0.5M，所述3,3'，5,5'-四甲基联苯胺溶液浓度范围为0.1～1.0mM，反应时间范围为5～30min。
本发明技术方案相对现有技术具有以下优点：
本发明技术方案基于抗原-抗体高度专一性反应以及铜-胺络合高效催化反应，可用于目视比色方法实现对外泌体溶液的高灵敏度检测，并且在没有任何仪器的帮助下，即可方便地实现低至2.5×105个/mL外泌体溶液的高灵敏检测。因此相对现有技术，本发明技术方案能够简单便捷且反应迅速的通过标记蛋白和外泌体特异性结合的快速检测外泌体，可实现外泌体的快速、高灵敏度的目视检测。与此同时本发明技术方案的检测方法具有成本低廉、反应效率高以及稳定性极好等优点，能够在外泌体检测和疾病诊断，特别是癌症的检测中得到广泛应用。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图示出的结构获得其他的附图。
图1为本发明实施例1中对照组和含109个/mL的外泌体溶液反应后得到的滤膜发生颜色变化对比图；
图2为本发明实施例2中对照组和不同浓度的外泌体溶液反应后得到的滤膜发生颜色变化对比图；
图3为本发明实施例3中对照组和含2.5×105个/mL的外泌体溶液反应后得到的滤膜发生颜色变化对比图。
本发明目的的实现、功能特点及优点将结合实施例，参照附图做进一步说明。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
需要说明，若本发明实施例中有涉及方向性指示(诸如上、下、左、右、前、后……)，则该方向性指示仅用于解释在某一特定姿态(如附图所示)下各部件之间的相对位置关系、运动情况等，如果该特定姿态发生改变时，则该方向性指示也相应地随之改变。
另外，若本发明实施例中有涉及“第一”、“第二”等的描述，则该“第一”、“第二”等的描述仅用于描述目的，而不能理解为指示或暗示其相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。另外，各个实施例之间的技术方案可以相互结合，但是必须是以本领域普通技术人员能够实现为基础，当技术方案的结合出现相互矛盾或无法实现时应当认为这种技术方案的结合不存在，也不在本发明要求的保护范围之内。
本发明提出一种高灵敏度目视检测外泌体的方法。
实施例1
在本发明实施例中，通过将1mL浓度为1013个/mL的CuS纳米粒子溶液加入至20uL带有巯基的CD63适配体溶液中，而后逐渐加入2M的氯化钠溶液，使得体系溶液最终的氯化钠浓度为0.1M，反应8h后离心并洗涤得到带有CD63适配体的CuS纳米粒子。
取1mL浓度为109个/mL的外泌体溶液，向其中加入20uL带有CD63适配体的CuS纳米粒子，反应半小时后加入0.1％的十二烷基硫酸钠并将溶液滤过50nm的滤膜，用PBS洗涤三次，得到含有外泌体-CuS的滤膜，在滤膜上加入10uL浓度为1.0×10-3M AgNO3溶液反应2min。
将新配的3,3'，5,5'-四甲基联苯胺(TMB)溶液和5uL新配的10mol/L过氧化氢溶液溶于500uL的三乙胺盐酸盐溶液中，均匀混合后制得检测溶液。
将上述检测溶液加入至对照组(不含外泌体的对照溶液重复上述实验得到的滤膜)以及含109个/mL的外泌体滤膜上，静置5min，通过目视法观察检测滤膜颜色变化，对照组(不含外泌体的对照溶液重复上述实验得到的滤膜)和含109个/mL的外泌体滤膜的颜色变化如图1。如图1所示，含有外泌体的滤膜相较于不含外泌体的滤膜在颜色上有着非常显著差异。
实施例2
在本发明实施例中，通过将1mL浓度为1013个/mL的CuS纳米粒子溶液加入至20uL带有巯基的CD63适配体溶液中，反应8h后离心并洗涤得到带有CD63适配体的CuS纳米粒子。
分别取1mL浓度为5×107个/mL、108个/mL、5×108个/mL、1.0×109个/mL的四种外泌体溶液，分别向其中加入20uL带有CD63适配体的CuS纳米粒子，反应半小时后加入0.1％的十六烷基三甲基溴化铵并将溶液滤过50nm的滤膜，用PBS洗涤三次，得到含有外泌体-CuS的滤膜，在滤膜上加入10uL的浓度为5.0×10-4AgNO3，反应3min。
新配的3,3'，5,5'-四甲基联苯胺(TMB)溶液和5uL新配的10mol/L的过氧化氢溶液溶于500uL的三乙胺盐酸盐溶液中，均匀混合后制得检测溶液。
将上述检测溶液加入至对照组(不含外泌体的对照溶液重复上述实验得到的滤膜)以及含有不同外泌体浓度的滤膜(5×107个/mL、108个/mL、5×108个/mL、1.0×109个/mL)中，反应5min后，通过目视比色法检测滤膜的颜色变化如图2。如图2所示，随着样品溶液的外泌体浓度不断增大，滤膜颜色不断加深，从而可达到肉眼直接观察效果。
实施例3
在本发明实施例中，通过将50mL浓度为1013个/mL的CuS纳米粒子溶液加入至1mL带有巯基的CD63适配体溶液中，反应8h后离心并洗涤得到带有CD63适配体的CuS纳米粒子。
取200mL浓度为2.5×105个/mL的外泌体溶液，向其中加入40mL带有CD63适配体的CuS纳米粒子，反应半小时后加入0.3％的十二烷基硫酸钠并将溶液滤过50nm的滤膜，用PBS洗涤三次，得到含有外泌体-CuS的滤膜，在滤膜上加入10uL的浓度为5.0×10-4AgNO3，反应5min。
新配的3，3'，5，5'-四甲基联苯胺(TMB)溶液和5uL新配的10mol/L过氧化氢溶液溶于250uL的三乙胺盐酸盐溶液中，均匀混合后制得检测溶液。
将上述检测溶液加入至对照组(不含外泌体的对照溶液重复上述实验得到的滤膜)以及含有2.5×105个/mL的外泌体滤膜中反应5min后，通过目视比色法检测滤膜的颜色变化如图3。
实施例4
在本发明实施例中，通过将1mL浓度为1013个/mL的CuS纳米粒子溶液加入至20uL带有巯基的CD63适配体溶液中，而后逐渐加入2M的氯化钠溶液使得体系溶液最终的氯化钠浓度为0.1M，反应8h后离心并洗涤得到带有CD63适配体的CuS纳米粒子。
取5mL浓度为107个/mL的外泌体溶液，向其中加入100uL带有CD63适配体的CuS纳米粒子，反应半小时后加入0.5％的十二烷基硫酸钠并将溶液滤过50nm的滤膜，用PBS洗涤三次，得到含有外泌体-CuS的滤膜，在滤膜上加入10uL的浓度为1.0×10-3M AgNO3溶液反应5min。
将新配的3,3'，5,5'-四甲基联苯胺(TMB)溶液和10uL新配的10mol/L过氧化氢溶液溶于400uL的三乙胺盐酸盐溶液中，均匀混合后制得检测溶液。
将上述检测溶液加入至对照组(不含外泌体的对照溶液重复上述实验得到的滤膜)以及含107个/mL的外泌体滤膜上，静置5min，通过目视法观察对照组和实验组的滤膜颜色变化。
以上所述仅为本发明的优选实施例，并非因此限制本发明的专利范围，凡是在本发明的构思下，利用本发明说明书及附图内容所作的等效结构变换，或直接/间接运用在其他相关的技术领域均包括在本发明的专利保护范围内。
Claims (5)
1.一种高灵敏度目视检测外泌体的方法，其特征在于，包括以下步骤：
步骤S1：将CuS纳米粒子和带有巯基的外泌体表面膜蛋白适配体反应后得到CuS-膜蛋白适配体结合物，所述CuS纳米粒子的大小为10~40 nm；
步骤S2：将含有外泌体的溶液滤过200 nm的滤膜，并向外泌体溶液中加入CuS-膜蛋白适配体结合物反应后得到外泌体-CuS结合物；
步骤S3：将步骤S2得到的反应液加入表面活性剂并通过50 nm的滤膜，使用PBS冲洗滤膜得到含有外泌体-CuS结合物的滤膜；所述表面活性剂为十二烷基硫酸钠、十六烷基三甲基溴化铵、聚乙烯吡咯烷酮中的一种或多种，表面活性剂的浓度范围为0.1~2.0% ；
步骤S4：向步骤S3得到的含有外泌体-CuS结合物的滤膜加入一定量的AgNO3溶液，反应后加入三乙胺盐酸盐、过氧化氢和3,3'，5,5'-四甲基联苯胺混合溶液反应显色，通过目视法观察滤膜的颜色变化。
2.如权利要求1所述的方法，其特征在于，所述步骤S1中，所述带有巯基的外泌体表面膜蛋白适配体为CD63适配体、CD81适配体、CD9适配体、EpCAM适配体、HER2适配体、MUC1适配体中的一种或多种；所述CuS纳米粒子和带有巯基的外泌体表面膜蛋白适配体的反应时间为10~24h。
3.如权利要求1所述的方法，其特征在于，所述步骤S2的外泌体溶液和CuS-膜蛋白适配体结合物反应时间为0.5~10 h。
4.如权利要求1所述的方法，其特征在于，所述步骤S2的所述外泌体溶液为1 mL，在外泌体溶液浓度通过提高外泌体溶液体积而提高灵敏度。
5.如权利要求1所述的方法，其特征在于，所述步骤S4的所述AgNO3溶液浓度范围为10-5~10-3 M，体积范围为10~50 uL，步骤S4的所述的滤膜中物质和AgNO3反应时间范围为5~10min，所述三乙胺盐酸盐浓度为0.05~0.2 M、过氧化氢浓度范围为0.1~0.5 M，所述3,3'，5,5'-四甲基联苯胺溶液浓度范围为0.1~1.0 mM，反应时间范围为5~30 min。
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010797874.XA CN111896751B (zh) | 2020-08-10 | 2020-08-10 | 一种高灵敏度目视检测外泌体的方法 |
| US17/204,954 US20220042979A1 (en) | 2020-08-10 | 2021-03-18 | Biological nanoparticle detecting method with high sensitivity |
| EP21185369.2A EP3954996A1 (en) | 2020-08-10 | 2021-07-13 | Biological nanoparticle detecting method with high sensitivity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010797874.XA CN111896751B (zh) | 2020-08-10 | 2020-08-10 | 一种高灵敏度目视检测外泌体的方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111896751A CN111896751A (zh) | 2020-11-06 |
| CN111896751B true CN111896751B (zh) | 2023-10-03 |
Family
ID=73246836
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010797874.XA Active CN111896751B (zh) | 2020-08-10 | 2020-08-10 | 一种高灵敏度目视检测外泌体的方法 |
Country Status (3)
| Country | Link |
|---|---|
| US (1) | US20220042979A1 (zh) |
| EP (1) | EP3954996A1 (zh) |
| CN (1) | CN111896751B (zh) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109632765A (zh) * | 2019-01-28 | 2019-04-16 | 上海理工大学 | 一种外泌体表面蛋白检测方法 |
| CN110208259A (zh) * | 2019-06-27 | 2019-09-06 | 南京医科大学 | 一种快速检测亚纳摩尔级的金属铜离子的方法 |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1207569C (zh) * | 1997-08-04 | 2005-06-22 | 株式会社先端生命科学研究所 | 用于测定含病毒样品的方法、病毒测定方法和诊断试剂盒 |
| KR101404507B1 (ko) * | 2012-04-12 | 2014-06-10 | 한국과학기술원 | 다수의 박막 구조물의 조합을 이용한 미소입자 처리 장치 |
| US11243215B2 (en) * | 2016-01-14 | 2022-02-08 | The Regents Of The University Of California | 3D-exoquant method for the analysis of surface molecules and quantification of tissue-specific exosomes in biological fluids |
-
2020
- 2020-08-10 CN CN202010797874.XA patent/CN111896751B/zh active Active
-
2021
- 2021-03-18 US US17/204,954 patent/US20220042979A1/en active Pending
- 2021-07-13 EP EP21185369.2A patent/EP3954996A1/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109632765A (zh) * | 2019-01-28 | 2019-04-16 | 上海理工大学 | 一种外泌体表面蛋白检测方法 |
| CN110208259A (zh) * | 2019-06-27 | 2019-09-06 | 南京医科大学 | 一种快速检测亚纳摩尔级的金属铜离子的方法 |
Non-Patent Citations (2)
| Title |
|---|
| Nan Cheng等.Recent Advances in Biosensors for Detecting Cancer-Derived Exosomes.Trends in Biotechnology.2019,第1-19页. * |
| Shan Chen等.Triethylamine as a complexing reagent for highly efficient naked-eyes copper ions sensing - a new catalytic pathway for ultrasensitive detection.Sensors and Actuators: B. Chemical.2020,第1-25页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| US20220042979A1 (en) | 2022-02-10 |
| EP3954996A1 (en) | 2022-02-16 |
| CN111896751A (zh) | 2020-11-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zeng et al. | Rapid and reliable detection of alkaline phosphatase by a hot spots amplification strategy based on well-controlled assembly on single nanoparticle | |
| Rong et al. | Metal ions doped chitosan–poly (acrylic acid) nanospheres: Synthesis and their application in simultaneously electrochemical detection of four markers of pancreatic cancer | |
| JP3088789B2 (ja) | イオンのアッセイ法とその装置 | |
| CN106198478B (zh) | 基于量子点比率荧光的分子印迹聚合物检测米托蒽醌的方法 | |
| CN107543809B (zh) | 一种谷胱甘肽分子检测用试纸条及检测方法 | |
| US20230081589A1 (en) | Universal Aptamer-based Colloidal Gold Lateral Flow Test Strip for Detecting Small-molecule Substances | |
| CN101851677A (zh) | 用于检测Hg2+的核酸纳米金生物传感器 | |
| CN107841527A (zh) | 一种利用核酸适配体和磁性材料检测凝血酶的荧光检测方法 | |
| Dong et al. | Polymerizing dopamine onto Q-graphene scaffolds towards the fluorescent nanocomposites with high aqueous stability and enhanced fluorescence for the fluorescence analysis and imaging of copper ions | |
| Zhuo et al. | One-step hydrothermal synthesis of silver-doped carbon quantum dots for highly selective detection of uric acid | |
| CN115308169A (zh) | 一种基于硫量子点和铜纳米簇的比率型荧光探针及其应用 | |
| Wang et al. | Cu 2+ modulated DNA-templated silver nanoclusters as a turn-on fluorescence probe for the detection of quinolones | |
| Wang et al. | A sensing platform for on-site detection of glutathione S-transferase using oxidized Pi@ Ce-doped Zr-based metal-organic frameworks (MOFs) | |
| CN111896751B (zh) | 一种高灵敏度目视检测外泌体的方法 | |
| Wang et al. | High-sensitivity biosensor based on SERS integrated with dendrimer-assisted boronic acid-functionalized magnetic nanoparticles for IL-6 detection in human serum | |
| EP1542013B1 (en) | Chemiluminescence enhancer | |
| CN111521572A (zh) | 一种Fe2+和铜蓝蛋白快速检测方法及应用 | |
| CN112816701B (zh) | 胶体金侧流层析法快速检测蓖麻毒素的方法及胶体金侧流层析试剂盒 | |
| CN113402470B (zh) | 一种多通道可逆比色汞离子荧光探针、制备方法与应用 | |
| CN108519358A (zh) | 一种基于新型金纳米颗粒检测Hg2+的方法 | |
| Kim et al. | Masking agent-controlled discriminative Hg2+ and Cu2+ sensing by quinonediimine dye formation of aniline-functionalized silica nanoparticles | |
| CN108241012B (zh) | 基于石墨烯的血红蛋白传感器电极的制备方法及其应用 | |
| EP3726215A1 (en) | Silica nanoparticles for biomarker diagnosis and method for producing same | |
| CN115975206B (zh) | 一种杂化mof材料及其制备和应用 | |
| Zhang et al. | Electrochemical sensing platform for detection of heavy metal ions without electrochemical signal |
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 |