CN114713299A - 一种微流控芯片及外泌体检测方法 - Google Patents
一种微流控芯片及外泌体检测方法 Download PDFInfo
- Publication number
- CN114713299A CN114713299A CN202210005840.1A CN202210005840A CN114713299A CN 114713299 A CN114713299 A CN 114713299A CN 202210005840 A CN202210005840 A CN 202210005840A CN 114713299 A CN114713299 A CN 114713299A
- Authority
- CN
- China
- Prior art keywords
- sio
- microfluidic chip
- chip
- mixer
- exosome
- 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.)
- Granted
Links
- 210000001808 exosome Anatomy 0.000 title claims abstract description 72
- 238000001514 detection method Methods 0.000 title abstract description 28
- 229910052681 coesite Inorganic materials 0.000 claims abstract description 45
- 229910052906 cristobalite Inorganic materials 0.000 claims abstract description 45
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 45
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 45
- 229910052682 stishovite Inorganic materials 0.000 claims abstract description 45
- 229910052905 tridymite Inorganic materials 0.000 claims abstract description 45
- 238000000034 method Methods 0.000 claims abstract description 20
- 239000000084 colloidal system Substances 0.000 claims abstract description 13
- 238000001338 self-assembly Methods 0.000 claims abstract description 11
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N titanium dioxide Inorganic materials O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 claims abstract description 11
- 238000005516 engineering process Methods 0.000 claims abstract description 7
- 239000002105 nanoparticle Substances 0.000 claims abstract description 7
- 238000002347 injection Methods 0.000 claims abstract description 6
- 239000007924 injection Substances 0.000 claims abstract description 6
- 239000000758 substrate Substances 0.000 claims description 8
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical group N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 6
- GUBGYTABKSRVRQ-DCSYEGIMSA-N Beta-Lactose Chemical class OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-DCSYEGIMSA-N 0.000 claims description 5
- 102100027221 CD81 antigen Human genes 0.000 claims description 5
- 102000018651 Epithelial Cell Adhesion Molecule Human genes 0.000 claims description 5
- 108010066687 Epithelial Cell Adhesion Molecule Proteins 0.000 claims description 5
- 102100041003 Glutamate carboxypeptidase 2 Human genes 0.000 claims description 5
- 101000914479 Homo sapiens CD81 antigen Proteins 0.000 claims description 5
- 101000892862 Homo sapiens Glutamate carboxypeptidase 2 Proteins 0.000 claims description 5
- 238000007112 amidation reaction Methods 0.000 claims description 5
- 230000003321 amplification Effects 0.000 claims description 5
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 5
- 229920001481 poly(stearyl methacrylate) Polymers 0.000 claims description 5
- 239000000427 antigen Substances 0.000 claims description 4
- 102000036639 antigens Human genes 0.000 claims description 4
- 108091007433 antigens Proteins 0.000 claims description 4
- 230000002194 synthesizing effect Effects 0.000 claims description 4
- 229960002685 biotin Drugs 0.000 claims description 3
- 235000020958 biotin Nutrition 0.000 claims description 3
- 239000011616 biotin Substances 0.000 claims description 3
- 238000010226 confocal imaging Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 238000011895 specific detection Methods 0.000 claims description 3
- 102000005936 beta-Galactosidase Human genes 0.000 claims description 2
- 108010005774 beta-Galactosidase Proteins 0.000 claims description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 2
- 229930195724 β-lactose Natural products 0.000 claims description 2
- 102100021023 Gamma-glutamyl hydrolase Human genes 0.000 claims 2
- 108010062699 gamma-Glutamyl Hydrolase Proteins 0.000 claims 2
- 230000009435 amidation Effects 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 238000002174 soft lithography Methods 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 15
- 206010028980 Neoplasm Diseases 0.000 description 12
- 238000002965 ELISA Methods 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 9
- 238000005199 ultracentrifugation Methods 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 239000004205 dimethyl polysiloxane Substances 0.000 description 6
- 235000013870 dimethyl polysiloxane Nutrition 0.000 description 6
- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 description 6
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 description 6
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 241000179948 Sarcophaga beta Species 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 238000006911 enzymatic reaction Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- PVGATNRYUYNBHO-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 4-(2,5-dioxopyrrol-1-yl)butanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCN1C(=O)C=CC1=O PVGATNRYUYNBHO-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 239000010839 body fluid Substances 0.000 description 2
- 210000005252 bulbus oculi Anatomy 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 230000008020 evaporation Effects 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000009832 plasma treatment Methods 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000005728 strengthening Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 206010063045 Effusion Diseases 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 206010061309 Neoplasm progression Diseases 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 208000009956 adenocarcinoma Diseases 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000011953 bioanalysis Methods 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000005034 decoration Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000003125 immunofluorescent labeling Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000035992 intercellular communication Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 239000011343 solid material Substances 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- OGIDPMRJRNCKJF-UHFFFAOYSA-N titanium oxide Inorganic materials [Ti]=O OGIDPMRJRNCKJF-UHFFFAOYSA-N 0.000 description 1
- 230000005751 tumor progression Effects 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
-
- 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
- G01N33/53—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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
-
- 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
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Dispersion Chemistry (AREA)
- Clinical Laboratory Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nanotechnology (AREA)
- Zoology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种微流控芯片及外泌体检测方法，其微流控通道内嵌有3D‑SiO2或3D‑TiO2人字形混合器，所述3D‑SiO2或3D‑TiO2人字形混合器为SiO2或TiO2纳米颗粒注入到3D‑SiO2人字形混合器模板中构建而成，混合器嵌入微流控通道。本发明采用软光刻方法制备3D‑SiO2模具，注入SiO2通过胶体自组装技术可以快速批量生产3D‑SiO2混合器，并与顶层封盖密封形成闭合芯片。本发明用于检测外泌体的方法灵敏度高，检测限低；同时芯片制作简单，可批量生产，可以集合各种纳米粒子进行不同目标物的检测与应用。
Description
技术领域
本发明涉及微流控设备和外泌体检测技术领域，具体的说，涉及3D-SiO2自组装微流控芯片的制备和外泌体的超灵敏检测方法。
背景技术
外泌体是大多数细胞分泌的30-150纳米的生物胞外囊泡，在细胞间通讯、传播致病因子、诱导肿瘤发生等方面发挥重要作用。肿瘤来源的外泌体在体液中积累，包括血液、尿液和恶性积液。因此，靶向肿瘤外泌体为确定肿瘤的进展和转移提供了一种微创的方法。然而，在疾病的早期阶段，体液中肿瘤源性外泌体是罕见的，因此，迫切需要开发新的生物传感器在正常细胞相关外泌体存在的情况下对肿瘤外泌体进行超敏感分析。常规的外泌体检测包括超离心分离、免疫印迹定量和酶联免疫吸附试验(ELISA)。标准的超离心分离需要多次离心(>8小时)，而且该方法对样品要求高、收率低。
微流控技术在生物分析方面具有非凡的优势，它为目标分子和传感器之间的有效接触提供了较大的表面积，极大地提高了分析灵敏度和速度。微流控小型化分析降低了样品消耗，同时减小了器件的整体尺寸，但微量样品对检测要求更高，对表面检测目标更敏感。
因此如何提高目标物与芯片表面的结合效率是提高检测灵敏度的关键。目前开发的不同形状的混合器，例如人字形，Y字形由固体材料组成，不仅有效结合面积减少，而且由于固体的完全阻塞，流体在固液界面的滞留大大降低了结合效率。
发明内容
为了克服现有技术的不足，本发明的目的在于开发一种3D-SiO2多孔芯片，并用于检测外泌体。该检测方法检测灵敏度高，检测限低；同时制备芯片的方法简单，成本低廉，能够批量生产。
本发明提供一种微流控芯片，其微流控通道内嵌有3D-SiO2或3D-TiO2人字形混合器，所述3D-SiO2或3D-TiO2人字形混合器为SiO2或TiO2纳米颗粒注入到3D-SiO2人字形混合器模板中构建而成，混合器嵌入微流控通道。
进一步，所述微流控芯片的制备方法为：制作模具，将SiO2或TiO2注入模板，通过胶体自组装技术合成3D-SiO2混合器，并与顶层、盖板封闭形成芯片。
进一步，所述微流控芯片表面通过酰胺化反应修饰捕获抗体Tim-4抗体。
进一步，所述微流控芯片通入生物素化的三个特异性检测抗体：CD81, PSMA和EpCAM。
进一步，特异性抗体与外泌体表面抗原结合，然后通入链霉亲和素修饰的β牛乳糖共轭酶及其底物β-半乳糖苷酶的底物荧光素2-β-D-吡喃半乳糖苷,β牛乳糖共轭酶与三个特异性抗体的生物素特异性结合，催化FDG底物进行荧光信号放大，通过共聚焦成像获得荧光强度对外泌体进行定量。
此外，本发明还提供一种外泌体的检测方法，使用权利要求1-5任意一项所述的微流控芯片。
具体的，本发明采用胶体自组装技术(CSA)为在微流控芯片中创建三维和周期性结构的传统技术提供了一种简单而廉价的替代方案。将具有良好生物相容性的SiO2纳米颗粒注入到3D-SiO2人字混合器模板中构建混合器，并将混合器嵌入微流控通道中，构建3D-SiO2多孔芯片。
通过传统软光刻的方法制作PDMS模具(其他本领域通用材料的模具也可以)，将SiO2单分散胶体溶液注入模板，通过胶体自组装技术合成3D-SiO2混合器，并与顶层盖板封闭形成芯片。
为了进一步提高外泌体检测的灵敏度，我们采用三种特异的外泌体标记物结合酶促反应来扩大信号。
首先在芯片表面通过酰胺化反应修饰捕获抗体Tim-4抗体，通入外泌体溶液后，外泌体被捕获在芯片表面，然后通入生物素化的三个特异性检测抗体：CD81, PSMA和EpCAM，特异性抗体与外泌体表面抗原结合，然后通入链霉亲和素修饰的β牛乳糖共轭酶(SβG)和SβG的底物FDG,SβG酶与三个特异性抗体的生物素特异性结合，催化FDG底物进行荧光信号放大，通过共聚焦成像获得荧光强度对外泌体进行定量。
本发明中，胶体自组装合成3D-SiO2混合器的具体步骤为：50μL单分散的 SiO2胶体溶液超声15分钟后，注入PDMS模具溶液储存槽，溶液自发地充满通道，并在通道出口处停止。所有通道填满后，用PDMS片覆盖储存槽，只留下出口处不封闭，通过胶体蒸发在通道中诱导胶体自组装生长。然后用5％的3-MPS 取代溶液，阻止胶体生长，同时增强SiO2人字结构的稳定性，加热处理(65℃， 1h)进一步增强3D-SiO2的机械性，揭开表面模具，对3D-SiO2混合器和上层封盖进行表面等离子体处理后，键合这两部分组装成3D-SiO2多孔芯片。
本发明中，酰胺化反应修饰捕获抗体Tim-4抗体具体步骤为：40μL马来酰亚胺基丁酰-氧琥珀酰亚胺脂(GMBS)溶液(0.3mg/mL)流过芯片进行抗体固定化，用20μL PBS洗涤三次后加入40μL SA(0.1mg/mL)反应1h,PBS洗涤三次，并将40μL生物素化Tim-4抗体(0.1mg/mL)充满芯片反应1h。芯片使用前用5％BSA封闭2h,4℃保存。
本发明中，三种特异性标记物联合酶促反应放大信号检测外泌体的具体步骤如下。用微注射器将40μL标准外泌体以0.5μL/min的速度泵入芯片，外泌体被包覆在芯片表面的Tim-4捕获。用1μL/min的PBS冲洗未结合的外泌体20min，并以0.5μL/min的速度注入三种生物素化抗体混合物。这三种生物素化抗体(各 20μg/mL)特异性识别外泌体上CD81、PSMA和EpCAM抗原，芯片用20μL PBS 清洗三次，然后0.5μL/min加入SβG酶(50ng/mL)30分钟。过剩的SβG用PBS 冲洗三次后，20μL FDG(500μM)加入芯片在黑暗中反应30分钟。在激光共聚焦下获得荧光图像，随机取5张图像通过ImageJ计算总荧光强度，定量外泌体。激光共聚焦拍摄图片时，激发波长：488nm，发射波长：514nm。
和现有技术相比，本发明的有益效果在于：
1.传统的芯片内部修饰是具有一定高度的PDMS实体柱，本发明在芯片内部构建多孔3D-SiO2，纳米粒子之间有序排列形成的小孔允许外泌体通过，可以减小液固界面流动阻力，大大提高了芯片表面与外泌体的结合效率，检测灵敏度提高，检测限为每微升220个外泌体。
2.通过胶体自组装技术合成3D-SiO2混合器，方法简单，可以批量生产。同时可以针对不同的检测物结合不同纳米材料构建混合器，例如肿瘤细胞大小是微米级，可以采用不同尺寸的TiO2纳米粒子，以期望形成微米尺寸的小孔进行实验。此方法灵活性强，应用广泛。
3.相较于传统使用单一抗体检测外泌体，本发明采用三种特异性标记物可以检测到外泌体上更多的蛋白，并联合酶促反应放大检测外泌体信号，特异性强，检测更灵敏。
4.本发明在基础实验中得到证实，并成功应用到临床前线腺癌病人血清外泌体检测中，
结果与商用酶联免疫吸附实验(ELISA)和免疫荧光染色结果一致。
附图说明
图1是3D-SiO2多孔芯片示意图：最下面一层是玻璃片，通过CSA胶体自组装在玻璃片上形成人字形的3D-SiO2，最后封闭上顶层盖片，形成闭合的微流控芯片。
图2是3D-SiO2多孔芯片用于外泌体捕获。(a)BSA包裹芯片捕获外泌体情况。(b)Tim-4抗体包裹芯片捕获外泌体。(c)Tim-4抗体包裹芯片捕获PBS(不含外泌体)。(d)外泌体溶液流经3D-SiO2多孔芯片前后的荧光强度变化。(e)3D-SiO2多孔芯片和超速离心方法(UC)的捕获效率比较。(f)荧光外泌体分布的3D图像。所有实验均平行三次，误差条代表三次测量的标准差。
图3是外泌体检测的最佳条件。(a)不同SβG浓度的荧光信号。(b)FDG孵育不同时间的荧光信号。(c)超速离心(UC)纯化的LNCaP和Vcap外泌体(106/μL) 与含或不含SβG的FDG底物孵育。(d)不同浓度的外泌体在两种方法中的响应荧光信号(6.2×100-6.2×109/mL)。(e)溶于PBS或血清中不同浓度外泌体与响应荧光信号之间的线性关系。(f)三个标记物对健康对照组和PCa患者的特异性。所有实验均平行三次，误差条表示基于三个重复实验的标准差。
图4是动物实验和临床应用。(a)不同时期Vcap肿瘤小鼠的生长情况。(b) 小鼠接种肿瘤细胞第25天和第55天从小鼠眼球中获得血液。(c)3D-SiO2多孔芯片在小鼠不同时期检测到的三个外泌体标记物表达。(d)比较标准ELISA和 3D-SiO2多孔芯片方法测定的临床血清样本中的三个外泌体标记物蛋白含量。(e) 与患者匹配的肿瘤组织的典型免疫荧光染色图像(患者5)。
具体实施方式
下面结合附图和实施例对本发明的技术方案进行详细说明。
以下实施例中单分散的SiO2购买于美国Bangs Laboratories公司，商用酶联免疫吸附实验(ELISA)购于上海联硕生物科技有限公司，其他未特别说明的均为市场上常规试剂、材料或仪器。
实施例一、3D-SiO2多孔芯片用于外泌体捕获。
1.胶体自组装技术合成3D-SiO2芯片。50μL单分散的SiO2胶体溶液超声15分钟后，注入PDMS模具溶液储存槽，溶液自发地充满通道，并在通道出口处停止。所有通道填满后，用PDMS片覆盖储存槽，只留下出口处不封闭，通过胶体蒸发在通道中诱导胶体自组装生长。然后用5％的3-MPS取代溶液，阻止胶体生长，同时增强SiO2人字结构的稳定性，加热处理(65℃，1h)进一步增强3D-SiO2的机械性，揭开表面模具，对3D-SiO2混合器和上层封盖进行表面等离子体处理后，键合这两部分组装成3D-SiO2多孔芯片。
图1是3D-SiO2多孔芯片的结构示意图：最下面一层是玻璃片(图中左下方所示)，通过CSA胶体自组装在玻璃片上形成人字形的3D-SiO2，最后封闭上顶层盖片(图中右上方所示)，形成闭合的微流控芯片。
2.通过酰胺化反应对芯片表面进行抗体Tim-4修饰。40μL马来酰亚胺基丁酰-氧琥珀酰亚胺脂(GMBS)溶液(0.3mg/mL)流过芯片进行抗体固定化，用20μL PBS洗涤三次后加入40μL SA(0.1mg/mL)反应1h,PBS洗涤三次，并将40μL 生物素化Tim-4抗体(0.1mg/mL)充满芯片并反应1h。芯片使用前用5％BSA 封闭2h,4℃保存。
3.芯片特异性比较。通过BSA包裹芯片，Tim-4抗体包裹芯片捕获外泌体以及 Tim-4抗体包裹芯片捕获PBS(不含外泌体)的对比实验，结果如图2(a)(b)(c) 所示。为了量化3D-SiO2多孔芯片的捕获效率，将PKH67染色外泌体加入PBS 或10倍稀释的健康血清(105/μL)中，注射到Tim-4包裹的芯片和对照芯片中。每 15分钟采集2μL溶液，用微荧光仪测量荧光，比较样品注入芯片前后的荧光强度(FL)，以确定外泌体捕获，结果如图2(d)所示。3D-SiO2多孔芯片的捕获效率与超速离心方法的捕获效率对比如图2(e)所示。荧光外泌体分布的3D图像如图2(f)所示。
实施例二、外泌体检测。
1.检测条件优化。不同浓度的SβG(5-5000ng/mL)的荧光响应信号如图3(a) 所示,FDG底物与酶反应时间如图3(b)所示。是否使用SβG放大荧光信号如图 3(c)所示。
2.外泌体检测。不同浓度的外泌体使用两种方法进行检测，检测结果如图3(d) 所示，传统固体芯片的理论检测限(LOD)为1.56×103/μL，而3D-SiO2多孔芯片的理论检测限(LOD)为2.2×102/μL(基于平均空白信号加3倍标准偏差)。UC纯化 LNCaP外泌体在PBS和10倍稀释的健康血清中的检测线性曲线如图3(e)所示。溶于PBS的LNCaP外泌体与荧光信号的线性相关方程为y＝0.5007logX-2.042, 范围为105-109/μL(R2＝0.9935)。溶于血清LNCaP外泌体浓度与荧光信号的线性相关方程为y＝0.4803logX-2.053,范围为105-109/μL(R2＝0.9883)。为了进一步证实CD81、PSMA和EpCAM外泌体标记物在前列腺癌病人血清中的特异性，我们使用单个标记和这三个标记的混合进行外泌体检测，结果如图3(f)所示。实施例三、外泌体检测用于动物实验和临床应用
1.小鼠实验。为了验证该方法的可行性，我们将3D-SiO2多孔芯片应用于荷瘤小鼠的诊断。将小鼠分为对照组(8只)和实验组(15只)。用照片记录Vcap肿瘤小鼠的生长情况，如图4(a)所示，在25天和55天从小鼠眼球中提取血液如图4(b) 所示。3D-SiO2多孔芯片检测的性能优于商业ELISA，如图4(c)所示。3D-SiO2多孔芯片检测在区分对照、早期PCa和晚期PCa方面优于单独标记物检测。
2.临床血清实验。选取20例PCa患者和10例健康个体的血清中进行了三种特异性标记物的外泌体分析。标准ELISA方法和3D-SiO2芯片对同一样品的测量结果如图4(d)所示。两种方法均能显著区分PCa患者和健康对照组，并且三个外泌体标记蛋白水平在PCa患者中表达高于对照组。为了测试外泌体是否反映了PCa肿瘤起源的分子特征，我们在20例患者匹配的肿瘤组织中进行了免疫荧光组织学研究。选取患者5号作为代表，图4(e)验证了三种外泌体标记在此患者组织中的表达。结合该患者的外泌体检测结果与组织学结果表明外泌体的标记水平与实体肿瘤之间存在相关性，这支持了外泌体作为肿瘤非侵入性替代活检的潜在应用。
Claims (6)
1.一种微流控芯片，其特征在于，所述微流控芯片的微流控通道内嵌有3D-SiO2或3D-TiO2人字形混合器，所述3D-SiO2或3D-TiO2人字形混合器为SiO2或TiO2纳米颗粒注入到3D-SiO2人字形混合器模板中构建而成，混合器嵌入微流控通道。
2.根据权利要求1所述的微流控芯片，其特征在于，所述微流控芯片的制备方法为：制作模具，将SiO2或TiO2注入模板，通过胶体自组装技术合成3D-SiO2混合器，并与顶层、盖板封闭形成芯片。
3.根据权利要求1所述的微流控芯片，其特征在于，所述微流控芯片表面通过酰胺化反应修饰捕获抗体Tim-4抗体。
4.根据权利要求3所述的微流控芯片，其特征在于，所述微流控芯片通入生物素化的三个特异性检测抗体：CD81,PSMA和EpCAM。
5.根据权利要求4所述的微流控芯片，其特征在于，特异性抗体与外泌体表面抗原结合，然后通入链霉亲和素修饰的β牛乳糖共轭酶及其底物β-半乳糖苷酶的底物荧光素2-β-D-吡喃半乳糖苷,β牛乳糖共轭酶与三个特异性抗体的生物素特异性结合，催化FDG底物进行荧光信号放大，通过共聚焦成像获得荧光强度对外泌体进行定量。
6.外泌体的检测方法，其特征在于，所述检测方法使用权利要求1-5任意一项所述的微流控芯片。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210005840.1A CN114713299B (zh) | 2022-01-05 | 2022-01-05 | 一种微流控芯片及外泌体检测方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210005840.1A CN114713299B (zh) | 2022-01-05 | 2022-01-05 | 一种微流控芯片及外泌体检测方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114713299A true CN114713299A (zh) | 2022-07-08 |
| CN114713299B CN114713299B (zh) | 2024-01-26 |
Family
ID=82236168
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210005840.1A Active CN114713299B (zh) | 2022-01-05 | 2022-01-05 | 一种微流控芯片及外泌体检测方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114713299B (zh) |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1648658A (zh) * | 2004-12-15 | 2005-08-03 | 南京大学 | 微流控芯片中微混合器和微反应器的快速制作方法 |
| US20080305011A1 (en) * | 2007-06-07 | 2008-12-11 | Samsung Electronics Co., Ltd. | Microfluidic chip and method of fabricating the same |
| US20180100853A1 (en) * | 2016-10-09 | 2018-04-12 | The University Of Kansas | Graphene oxide-based nanolab and methods of detecting of exosomes |
| CN110055158A (zh) * | 2019-04-09 | 2019-07-26 | 厦门大学 | 一种微流控芯片的动态修饰方法及其捕获CTCs的应用 |
| CN110320355A (zh) * | 2018-03-30 | 2019-10-11 | 上海市肿瘤研究所 | 一种微流控芯片及循环外泌体的检测方法 |
| CN110361370A (zh) * | 2019-07-31 | 2019-10-22 | 深圳中山泌尿外科医院 | 一种基于微流控液滴的单胚胎分泌蛋白定量检测方法 |
| CN111304054A (zh) * | 2020-02-28 | 2020-06-19 | 重庆医科大学附属第三医院(捷尔医院） | 外泌体分离微流控芯片及其方法 |
| CN111505309A (zh) * | 2020-04-24 | 2020-08-07 | 东南大学 | 外泌体表面蛋白及内部miRNA同时检测芯片和检测技术 |
-
2022
- 2022-01-05 CN CN202210005840.1A patent/CN114713299B/zh active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1648658A (zh) * | 2004-12-15 | 2005-08-03 | 南京大学 | 微流控芯片中微混合器和微反应器的快速制作方法 |
| US20080305011A1 (en) * | 2007-06-07 | 2008-12-11 | Samsung Electronics Co., Ltd. | Microfluidic chip and method of fabricating the same |
| US20180100853A1 (en) * | 2016-10-09 | 2018-04-12 | The University Of Kansas | Graphene oxide-based nanolab and methods of detecting of exosomes |
| CN110320355A (zh) * | 2018-03-30 | 2019-10-11 | 上海市肿瘤研究所 | 一种微流控芯片及循环外泌体的检测方法 |
| CN110055158A (zh) * | 2019-04-09 | 2019-07-26 | 厦门大学 | 一种微流控芯片的动态修饰方法及其捕获CTCs的应用 |
| CN110361370A (zh) * | 2019-07-31 | 2019-10-22 | 深圳中山泌尿外科医院 | 一种基于微流控液滴的单胚胎分泌蛋白定量检测方法 |
| CN111304054A (zh) * | 2020-02-28 | 2020-06-19 | 重庆医科大学附属第三医院(捷尔医院） | 外泌体分离微流控芯片及其方法 |
| CN111505309A (zh) * | 2020-04-24 | 2020-08-07 | 东南大学 | 外泌体表面蛋白及内部miRNA同时检测芯片和检测技术 |
Non-Patent Citations (1)
| Title |
|---|
| XIANYING LI等: "Modified micro-space using self-organized nanoparticles for reduction of methylene blue", 《CHEMICAL COMMUNICATIONS》, pages 964 - 965 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114713299B (zh) | 2024-01-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ye et al. | Advances in nanotechnology for cancer biomarkers | |
| Li et al. | Emerging nanotechnologies for liquid biopsy: the detection of circulating tumor cells and extracellular vesicles | |
| Gaikwad et al. | Advances in point-of-care diagnostic devices in cancers | |
| Perfézou et al. | Cancer detection using nanoparticle-based sensors | |
| Ravalli et al. | Gold and magnetic nanoparticles-based electrochemical biosensors for cancer biomarker determination | |
| Otieno et al. | On-line protein capture on magnetic beads for ultrasensitive microfluidic immunoassays of cancer biomarkers | |
| CN103116023B (zh) | 用于检测肿瘤标志物的电化学发光免疫传感器及其制备方法和应用 | |
| Xie et al. | A novel electrochemical microfluidic chip combined with multiple biomarkers for early diagnosis of gastric cancer | |
| CN109490528A (zh) | 外泌体分析及癌症诊断方法 | |
| US20120100560A1 (en) | Device for capture, enumeration, and profiling of circulating tumor cells | |
| Vandghanooni et al. | Recent advances in aptamer-based nanosystems and microfluidics devices for the detection of ovarian cancer biomarkers | |
| Sharma et al. | Advances in ovarian cancer diagnosis: A journey from immunoassays to immunosensors | |
| CN109321577A (zh) | 一种检测外泌体的适配体组、侧流式适配体生物传感器及其制备方法 | |
| Ahirwar | Recent advances in nanomaterials-based electrochemical immunosensors and aptasensors for HER2 assessment in breast cancer | |
| Zamay et al. | Aptamer-based methods for detection of circulating tumor cells and their potential for personalized diagnostics | |
| Kavetskyy et al. | Magneto-immunoassay of cancer biomarkers: Recent progress and challenges in biomedical analysis | |
| Nakhjavani et al. | Biosensors for prostate cancer detection | |
| WO2018171318A1 (zh) | 一种用于全血中循环肿瘤细胞富集和检测的二氧化硅纳米线阵列芯片及制备方法 | |
| Maia et al. | Finding the perfect match between nanoparticles and microfluidics to respond to cancer challenges | |
| Ma et al. | Progress in nanomaterials-based optical and electrochemical methods for the assays of exosomes | |
| Karimzadeh et al. | Bio-assay: The best alternative for conventional methods in detection of epidermal growth factor | |
| Chen et al. | Colorimetric biosensing assays based on gold nanoparticles functionalized/combined with non-antibody recognition elements | |
| CN114713299A (zh) | 一种微流控芯片及外泌体检测方法 | |
| KR101612094B1 (ko) | 표적-특이적 프로브와 표지물질-항체 복합체를 포함하는 바이오마커 탐지용 조성물 및 이의 이용방법 | |
| KR20140008608A (ko) | 입자 복합체 및 이를 이용한 표적 세포 분리 방법 |
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 |