CN113552345A - 一种基于免疫荧光增强的外泌体定量检测方法 - Google Patents
一种基于免疫荧光增强的外泌体定量检测方法 Download PDFInfo
- Publication number
- CN113552345A CN113552345A CN202110648316.1A CN202110648316A CN113552345A CN 113552345 A CN113552345 A CN 113552345A CN 202110648316 A CN202110648316 A CN 202110648316A CN 113552345 A CN113552345 A CN 113552345A
- Authority
- CN
- China
- Prior art keywords
- antibody
- exosome
- exosomes
- quantitative detection
- film
- 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 93
- 238000001514 detection method Methods 0.000 title claims abstract description 37
- 238000010166 immunofluorescence Methods 0.000 title claims abstract description 19
- 238000000034 method Methods 0.000 claims abstract description 21
- 229910052751 metal Inorganic materials 0.000 claims abstract description 17
- 239000002184 metal Substances 0.000 claims abstract description 17
- 210000001124 body fluid Anatomy 0.000 claims abstract description 15
- 239000010839 body fluid Substances 0.000 claims abstract description 15
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 13
- 239000002082 metal nanoparticle Substances 0.000 claims abstract description 11
- 239000002120 nanofilm Substances 0.000 claims abstract description 10
- 238000007865 diluting Methods 0.000 claims abstract description 4
- 238000000799 fluorescence microscopy Methods 0.000 claims abstract description 4
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 34
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims description 24
- 239000003550 marker Substances 0.000 claims description 16
- 102000004169 proteins and genes Human genes 0.000 claims description 15
- 108090000623 proteins and genes Proteins 0.000 claims description 15
- 239000004038 photonic crystal Substances 0.000 claims description 12
- 239000010931 gold Substances 0.000 claims description 10
- 229910052737 gold Inorganic materials 0.000 claims description 10
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- 239000002073 nanorod Substances 0.000 claims description 6
- 239000002131 composite material Substances 0.000 claims description 5
- 239000000975 dye Substances 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 abstract description 6
- 210000004369 blood Anatomy 0.000 abstract description 4
- 239000008280 blood Substances 0.000 abstract description 4
- 238000001917 fluorescence detection Methods 0.000 abstract description 2
- 239000010408 film Substances 0.000 description 32
- 239000000758 substrate Substances 0.000 description 32
- 239000000243 solution Substances 0.000 description 25
- 239000011521 glass Substances 0.000 description 15
- 210000002381 plasma Anatomy 0.000 description 12
- 101000934368 Homo sapiens CD63 antigen Proteins 0.000 description 11
- 102100025222 CD63 antigen Human genes 0.000 description 10
- -1 CD31 Proteins 0.000 description 9
- 208000019425 cirrhosis of liver Diseases 0.000 description 8
- 229910052709 silver Inorganic materials 0.000 description 8
- 239000004332 silver Substances 0.000 description 8
- 201000007270 liver cancer Diseases 0.000 description 7
- 208000014018 liver neoplasm Diseases 0.000 description 7
- 239000008055 phosphate buffer solution Substances 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000002493 microarray Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 238000005406 washing Methods 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 239000012091 fetal bovine serum Substances 0.000 description 4
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(I) nitrate Inorganic materials [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 4
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 3
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 238000010926 purge Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- GWOLZNVIRIHJHB-UHFFFAOYSA-N 11-mercaptoundecanoic acid Chemical compound OC(=O)CCCCCCCCCCS GWOLZNVIRIHJHB-UHFFFAOYSA-N 0.000 description 2
- 102000000412 Annexin Human genes 0.000 description 2
- 108050008874 Annexin Proteins 0.000 description 2
- 102100034283 Annexin A5 Human genes 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 102100032912 CD44 antigen Human genes 0.000 description 2
- 102100027221 CD81 antigen Human genes 0.000 description 2
- 102100037904 CD9 antigen Human genes 0.000 description 2
- 101710163595 Chaperone protein DnaK Proteins 0.000 description 2
- 102000002029 Claudin Human genes 0.000 description 2
- 108050009302 Claudin Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 102000018651 Epithelial Cell Adhesion Molecule Human genes 0.000 description 2
- 108010066687 Epithelial Cell Adhesion Molecule Proteins 0.000 description 2
- 108010074556 Golgin subfamily A member 2 Proteins 0.000 description 2
- 229910004042 HAuCl4 Inorganic materials 0.000 description 2
- 102100028967 HLA class I histocompatibility antigen, alpha chain G Human genes 0.000 description 2
- 108010024164 HLA-G Antigens Proteins 0.000 description 2
- 101710178376 Heat shock 70 kDa protein Proteins 0.000 description 2
- 101710152018 Heat shock cognate 70 kDa protein Proteins 0.000 description 2
- 101710113864 Heat shock protein 90 Proteins 0.000 description 2
- 102100034051 Heat shock protein HSP 90-alpha Human genes 0.000 description 2
- 101000780122 Homo sapiens Annexin A5 Proteins 0.000 description 2
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 2
- 101000914479 Homo sapiens CD81 antigen Proteins 0.000 description 2
- 101000738354 Homo sapiens CD9 antigen Proteins 0.000 description 2
- 101001051490 Homo sapiens Neural cell adhesion molecule L1 Proteins 0.000 description 2
- 101000613251 Homo sapiens Tumor susceptibility gene 101 protein Proteins 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 108010064593 Intercellular Adhesion Molecule-1 Proteins 0.000 description 2
- 102100037877 Intercellular adhesion molecule 1 Human genes 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 102100024964 Neural cell adhesion molecule L1 Human genes 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 description 2
- 102100021688 Rho guanine nucleotide exchange factor 5 Human genes 0.000 description 2
- FOIXSVOLVBLSDH-UHFFFAOYSA-N Silver ion Chemical compound [Ag+] FOIXSVOLVBLSDH-UHFFFAOYSA-N 0.000 description 2
- 102100030552 Synaptosomal-associated protein 25 Human genes 0.000 description 2
- 101710149792 Triosephosphate isomerase, chloroplastic Proteins 0.000 description 2
- 101710195516 Triosephosphate isomerase, glycosomal Proteins 0.000 description 2
- 102100040879 Tumor susceptibility gene 101 protein Human genes 0.000 description 2
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 210000003722 extracellular fluid Anatomy 0.000 description 2
- 238000002073 fluorescence micrograph Methods 0.000 description 2
- 238000007306 functionalization reaction Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 102000006495 integrins Human genes 0.000 description 2
- 108010044426 integrins Proteins 0.000 description 2
- 239000004005 microsphere Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 239000002105 nanoparticle Substances 0.000 description 2
- 108010032037 rab5 GTP-Binding Proteins Proteins 0.000 description 2
- 102000007575 rab5 GTP-Binding Proteins Human genes 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 108040000979 soluble NSF attachment protein activity proteins Proteins 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000010409 thin film Substances 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- FFRBMBIXVSCUFS-UHFFFAOYSA-N 2,4-dinitro-1-naphthol Chemical compound C1=CC=C2C(O)=C([N+]([O-])=O)C=C([N+]([O-])=O)C2=C1 FFRBMBIXVSCUFS-UHFFFAOYSA-N 0.000 description 1
- 101710134784 Agnoprotein Proteins 0.000 description 1
- 101100165655 Arabidopsis thaliana BRO1 gene Proteins 0.000 description 1
- OPXJNKGZONUZHL-UHFFFAOYSA-N C1(=CC=CC=C1)C=C.C(C)(C)C(C(=O)N)=C Chemical compound C1(=CC=CC=C1)C=C.C(C)(C)C(C(=O)N)=C OPXJNKGZONUZHL-UHFFFAOYSA-N 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241000252506 Characiformes Species 0.000 description 1
- 101100120289 Drosophila melanogaster Flo1 gene Proteins 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 206010019668 Hepatic fibrosis Diseases 0.000 description 1
- 101100082597 Homo sapiens PDCD6IP gene Proteins 0.000 description 1
- 238000004566 IR spectroscopy Methods 0.000 description 1
- 239000011837 N,N-methylenebisacrylamide Substances 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 102100033344 Programmed cell death 6-interacting protein Human genes 0.000 description 1
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical group C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 239000000908 ammonium hydroxide Substances 0.000 description 1
- 229910001870 ammonium persulfate Inorganic materials 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- GRWVQDDAKZFPFI-UHFFFAOYSA-H chromium(III) sulfate Chemical compound [Cr+3].[Cr+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O GRWVQDDAKZFPFI-UHFFFAOYSA-H 0.000 description 1
- 230000003749 cleanliness Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 102000057640 human CD63 Human genes 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 210000002487 multivesicular body Anatomy 0.000 description 1
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 description 1
- QNILTEGFHQSKFF-UHFFFAOYSA-N n-propan-2-ylprop-2-enamide Chemical compound CC(C)NC(=O)C=C QNILTEGFHQSKFF-UHFFFAOYSA-N 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000003169 placental effect Effects 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Images
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
- G01N33/569—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
- G01N33/543—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
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
Abstract
本发明公开一种基于免疫荧光增强的外泌体定量检测方法。方法包括步骤:采用离心法提取外泌体或将待测体液如血液稀释;将金属纳米粒子荧光探针与外泌体或稀释后的待测体液结合,然后置于金属纳米膜上进行荧光成像,实现外泌体或待测体液中外泌体定量检测。本发明建立基于免疫增强的荧光检测方法,获得外泌体或特异性来源外泌体的增强荧光信号,建立标准曲线,获得待检测外泌体的浓度,能够满足外泌体及不同外泌体亚群的实验室及临床定量检测需求。
Description
技术领域
本发明涉及生物医学领域,尤其涉及一种基于免疫荧光增强的外泌体定量检测方法。
背景技术
外泌体(exosomes)是由细胞内多泡体与细胞膜融合后释放到细胞外基质中直径为30-100nm膜性小囊泡,内容物丰富,携带有蛋白、脂质和miRNA等。外泌体可由机体多种类型细胞释放,广泛分布于血液、尿液、唾液、乳汁、组织液等体液中。作为一种分子媒介,外泌体可以促进细胞间或器官间的信息交流,参与多种生理病理过程,与疾病的发生、发展和进程密切相关。外泌体能反映来源细胞的功能,病理条件下,其内容物组分较健康状态有所不同。外泌体及不同来源的外泌体具有不同的特异性表面标志蛋白,如ALIX,Annexin,ANXA5,CD171,CD31,CD44,CD63,CD81,CD9,Claudins,EpCAM,FLOT1,GM130,HLA-G,HSP5,HSP70,HSP90,ICAM-1,Integrins,RAB5,SNAP,TIM,TSG101等。根据表面标志蛋白的不同,可以将外泌体分为不同亚群,或称为不同来源外泌体(如肿瘤来源、神经来源、胎盘来源等)。识别和定量外泌体及不同外泌体亚群,能够为疾病的预测、诊断,疗效评估及预后提供信息。而在评价外泌体(总外泌体、不同亚群外泌体)分离纯化方法和分离效果时,亦需对分离到的外泌体进行定量。
用于外泌体定量检测的方法主要有酶联免疫吸附试验(ELISA)、免疫印迹(Western blot)、微流控、质谱等。外泌体尤其是不同亚群的外泌体,在体液中的含量低,目前尚缺乏快速、简便、灵敏度高、经济适用的检测方法。免疫荧光是生物医学检测中一种重要的方法,但现有方法存在荧光亮度不高,易于淬灭等缺点。
发明内容
鉴于上述现有技术的不足,本发明的目的在于提供一种基于免疫荧光增强的外泌体定量检测方法,旨在解决现有免疫荧光方法存在荧光亮度不高的问题。
本发明的技术方案如下:
一种基于免疫荧光增强的外泌体定量检测方法,其中,包括步骤:
采用离心法提取外泌体或将待测体液如血液稀释;
将金属纳米粒子荧光探针与外泌体或稀释后的待测体液结合,然后置于金属纳米膜上进行荧光成像,实现外泌体或待测体液中外泌体定量检测。
可选地,所述金属纳米粒子荧光探针由金属纳米棒(gold nanorods,GNRs)、外泌体表面标志蛋白抗体和荧光剂结合而成。
可选地,所述金属纳米棒为金纳米棒。
可选地,所述外泌体表面标志蛋白抗体选自抗ALIX抗体、抗Annexin抗体、抗ANXA5抗体、抗CD171抗体、抗CD31抗体、抗CD44抗体、抗CD63抗体、抗CD81抗体、抗CD9抗体、抗Claudins抗体、抗EpCAM抗体、抗FLOT1抗体、抗GM130抗体、抗HLA-G抗体、抗HSP5抗体、抗HSP70抗体、抗HSP90抗体、抗ICAM-1抗体、抗Integrins抗体、抗RAB5抗体、抗SNAP抗体、抗TIM抗体和抗TSG101抗体等抗体中的一种,包括单克隆或多克隆抗体。
可选地,所述荧光剂为AF647染料。
可选地,所述金属纳米膜为纳米银膜;
或者,所述金属纳米膜为纳米银膜和形成于所述纳米银膜上的PC668光子晶体膜构成的复合膜;
或者,所述金属纳米膜为纳米金膜。
有益效果:本发明建立基于免疫增强的荧光检测方法,获得外泌体或特异性来源外泌体的增强荧光信号,建立标准曲线,获得待检测外泌体的浓度,能够满足外泌体及不同外泌体亚群的实验室及临床定量检测需求。通过该方法,显著提高了外泌体定量检测的灵敏度。
附图说明
图1为金纳米棒包被抗体和“夹心法”免疫检测外泌体示意图。以CD63表面标记为例。
图2为用于荧光增强的GNRs/Ag(银)基底的表面形态特征;其中,a)为GNRs的透射电子显微镜图像。b)为GNRs(λ=645nm)和抗体包裹的GNRs(λ=662nm)溶液在紫外-红外光谱下的图像。c)为AF647分子分别在GNRs/Ag基底,GNRs,银岛膜和玻璃基底上的荧光图像。d)为c)中4种基底荧光增强因子的直方图。
图3为GNRs/Ag基底上的以人CD63表面标记外泌体为标准品的微阵列检测结果图。a)CD63表面标记外泌体被稀释成不同浓度进行检测,每个样品重复4次。图中左上为对照,其余的浓度依次为500-0.4ng/mL。b)CD63表面标记外泌体检测标准曲线。
图4为肝纤维化和肝癌患者血浆中外泌体(CD63表面标志)检测结果示意图。a)为扫描GNRs/Ag基底上带有AF647荧光标记抗体的微阵列生成的荧光图像,其中上面一排为2个对照的检测结果;病人1-3,肝纤维化患者;病人4-6,肝癌患者。b)为基于GNRs/Ag基底检测肝纤维化、肝癌患者血浆外泌体的荧光强度直方图,*p<0.05。
图5为AF647在不同基底上的荧光强度示意图;其中,a)为AF647分别在玻璃、Ag膜、PC668、Ag-PC668基底上的荧光强度示意图,b)为AF647分别在玻璃、Ag膜、PC668、Ag-PC668基底上的荧光强度直方图。
具体实施方式
本发明提供一种基于免疫荧光增强的外泌体定量检测方法,为使本发明的目的、技术方案及效果更加清楚、明确,以下对本发明进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
本发明提供一种基于免疫荧光增强的外泌体定量检测方法,其中,包括步骤:
采用离心法提取外泌体或将待测体液如血液稀释;
将金属纳米粒子荧光探针与外泌体或稀释后的待测体液结合,然后置于金属纳米膜上进行荧光成像,实现外泌体或待测体液中外泌体定量检测。
在一种实施方式中,所述金属纳米粒子荧光探针由金属纳米棒、外泌体表面标志蛋白抗体和荧光剂结合而成。进一步地,所述金属纳米棒为金纳米棒。进一步地,所述外泌体表面标志蛋白抗体选自抗ALIX抗体、抗Annexin抗体、抗ANXA5抗体、抗CD171抗体、抗CD31抗体、抗CD44抗体、抗CD63抗体、抗CD81抗体、抗CD9抗体、抗Claudins抗体、抗EpCAM抗体、抗FLOT1抗体、抗GM130抗体、抗HLA-G抗体、抗HSP5抗体、抗HSP70抗体、抗HSP90抗体、抗ICAM-1抗体、抗Integrins抗体、抗RAB5抗体、抗SNAP抗体、抗TIM抗体和抗TSG101抗体等抗体中的一种,包括单克隆或多克隆抗体。进一步地,所述荧光剂为AF647染料。
在一种实施方式中,所述金属纳米膜为纳米银膜或Ag-PC668或纳米金膜。
结合图1所示,本实施例提供一种基于免疫荧光增强的外泌体定量检测方法,具体基于银纳米粒子表面等离激元设计一种荧光增强结构,或基于银纳米粒子表面等离激元与胶体光子晶体相耦合原理设计一种荧光增强结构,即沉积在基板上的纳米银膜或光子晶体-纳米银复合膜(Ag-PC668);也可以基于金纳米粒子表面等离激元设计一种荧光增强结构,即沉积在基板上的纳米金膜。同时建立一种基于金纳米棒的夹心免疫测定法对外泌体进行定量检测。以外泌体表面标志蛋白抗体,如抗ALIX,Annexin,ANXA5,CD171,CD31,CD44,CD63,CD81,CD9,Claudins,EpCAM,FLOT1,GM130,HLA-G,HSP5,HSP70,HSP90,ICAM-1,Integrins,RAB5,SNAP,TIM,TSG101的单克隆或多克隆抗体等,包被金纳米棒。从体液(血浆、血清、尿液、组织液)、细胞、组织培养液中提取总外泌体,用包被了抗体的金纳米棒特异性捕获具有相应表面标志蛋白的外泌体或外泌体亚群。一旦抗体包裹的金纳米棒捕获到抗原,另一种类型的特异性一抗将用于结合抗原。带有荧光标记(AF647)的二抗用来识别用于结合抗原的一抗。将附着有荧光标记的金纳米棒悬浮液滴至纳米银膜或Ag-PC668或纳米金膜基底上,采用激光(如635nm激光)激发,在微阵列扫描仪中成像。以不同浓度的外泌体特异性表面标志蛋白或外泌体为标准品构建标准曲线进行检测,可获得相应的待检测外泌体的浓度。通过该方法,显著提高了外泌体定量检测的灵敏度,与玻璃基底相比,以纳米银膜为基底时,获得了360倍的增强,而以Ag-PC668或纳米金膜为基底时,将获得更高的增强倍数。且该方法一次可以同时检测4个以上病人,每个病人重复3次。
下面通过具体的实施例对本发明作进一步地说明。
实施例1
1、纳米银膜(银岛膜)的制备:
a.将清洗干净的载玻片放入配置好的piranha溶液(过氧化氢与浓硫酸按照体积比为1:3混合)中,浸泡8h。
b.将浸泡后的载玻片置于含45mL 0.03M AgNO3溶液的培养皿中,加入3mL氨水,在振荡器中振荡30min。
c.将经步骤b处理后的载玻片放入60mL 0.04M AgNO3溶液和30mL1.25M NaOH溶液混合的溶液中。加入12mL氨水将形成的沉淀物重新溶解。随后加入体积为30mL、浓度为0.25M的葡萄糖溶液,使银离子还原为银金属,在载玻片上形成银膜。20min后,从培养皿中取出载玻片,用超纯水冲洗,氮气吹干,密封避光保存。
2、金纳米棒的制备及功能化
a.金纳米棒的制备
在室温下通过两步种子介导生长法制备金纳米棒,具体步骤如下:
首先,将5mL 0.2M的CTAB(十六烷基三甲基溴化铵)溶液与5mL 5×10-4M的HAuCl4溶液混合,并轻轻搅拌后,加入新鲜制备的冰镇的0.6mL0.01M的NaBH4溶液(溶剂为水),搅拌2min,使溶液的颜色变成棕黄色,得到种子液。
然后,在5mL 0.2M的CTAB溶液中加入0.2mL 0.004M的AgNO3溶液,然后加入5mL0.001M的HAuCl4溶液,轻轻地混合,使得溶液变成金黄色。加入0.07mL 0.0788M的抗坏血酸溶液,摇匀,使溶液变为无色,得到生长液。
最后,在生长液中加入20μL种子液,室温过夜。当生长液呈深洋红色,表明金纳米棒成功形成。取上述过夜后的溶液1mL,以9600rpm的转速离心三次,每次20min。第三轮结束后,将金纳米棒沉淀置于水中使其再分散,以去除多余的CTAB和球形纳米颗粒。将金纳米棒沉淀分散于50μL的水中保存。
b.金纳米棒的功能化
在室温下将金纳米棒溶液浸泡在含20mM MUA(11-巯基十一烷酸)的乙醇中过夜。以14,000rpm离心12min,收集MUA修饰后的金纳米棒,用乙醇冲洗并干燥后,置于含20mM 1-乙基-3-(-3-二甲氨基丙基碳二酰亚胺盐酸盐(EDC)和20mM n-羟基琥珀酰亚胺(NHS)的甲醇溶液中浸泡30min以激活羧基,用于抗体偶联。
3、肝纤维化、肝癌患者血浆CD63表面标识外泌体的检测
a、标准曲线的绘制
羧基基团功能化的金纳米棒用于包被一抗(即兔抗外泌体表面特异性标志蛋白单克隆抗CD63抗体),将其置于质量浓度为15%FBS(胎牛血清)的PBS(磷酸盐缓冲液)中,1/100稀释,摇床中震荡2h。采用PBS洗涤3次。对照用纯FBS替代一抗。标准蛋白用含15%FBS的PBS稀释,最终浓度为0.4-500ng/mL。取50μL加入金纳米棒中,摇床中孵育2h,PBS洗涤金纳米棒3次。加入小鼠抗外泌体表面特异性标志蛋白单克隆抗体,室温,摇床中孵育1h。PBS洗涤3次,加入标记AF647的山羊抗小鼠抗体,室温,避光振荡孵育30min,PBS洗涤3次。将附着有荧光标记的金纳米棒悬浮液在纳米银膜或Ag-PC668基底或纳米金膜上滴干,并在635nm激光通道下在微阵列扫描仪中成像。获得发射光荧光强度后,绘制标准曲线。
图2为用于荧光增强的GNRs/Ag基底的表面形态特征。图2中a)展示了GNRs的尺寸均匀。图2中b)为GNRs(λ=645nm)和抗体包裹的GNRs(λ=662nm)溶液在紫外-红外光谱下的图像。图2中c)为AF647分子分别在GNRs/Ag基底,GNRs,银岛膜和玻璃上的荧光图像。图2中d)为c)中4种基底荧光增强因子的直方。
b、肝纤维化、肝癌患者血浆中外泌体的定量检测
利用离心的方法,从肝纤维化、肝癌患者的血浆中提取外泌体,或对人血浆样品进行稀释,用兔抗人抗CD63抗体包被金纳米棒,从外泌体或稀释的血浆中捕获待定量的外泌体,进行表面增强免疫荧光的检测,获得肿瘤患者血浆中外泌体较对照增强的荧光强度。通过标准曲线(图3),获得患者血浆外泌体的浓度。具体地,图4中a)为扫描GNRs/Ag基底上带有AF647荧光标记抗体的微阵列生成的荧光图像,其中上面一排为2个健康对照的检测结果;下面三排为病人的检测结果:病人1-3,肝纤维化患者;病人4-6,肝癌患者。图4中b)为基于GNRs/Ag基底检测的对照与病例(肝纤维化、肝癌患者)血浆外泌体的荧光强度直方图。以CD63作为标准品做标准曲线(图3),检测得到3个肝纤维化患者血浆中外泌体的浓度分别为16,24,28ng/mL,肝癌患者的浓度分别为34,44,54ng/mL。依据微阵列的通量不同,该方法一次至少可以同时检测4个或以上病人,且每个病人重复3次。
4、光子晶体与银纳米膜复合体的制备及不同基底荧光增强效果比较
a、p(St-co-NIPAM)(聚(苯乙烯-异丙基丙烯酰胺))微球的合成
将1.54g NIPAM和0.3g BIS(N,N-亚甲基双丙烯酰胺)单体溶解于95mL超纯水中,然后装入配有冷凝器、温度计和气体净化入口的三颈圆底烧瓶中。在搅拌条件下,将该烧瓶中溶液加热到70℃,同时使用氮气吹扫至少30min。在引发前约5min加入6.6g苯乙烯单体,并加入过硫酸铵,引发反应。氮气吹扫搅拌24h。使用Whatman滤纸(2号)过滤以除去凝结物,离心除去所有未反应的单体,得到粒径为668nm的光子晶体p(St-NIPAM)微球。
b、光子晶体薄膜的制备
首先用硫酸铬溶液处理玻璃基板以确保表面清洁,然后在恒定温度(50℃)和湿度(60%)下,将玻璃基板垂直放置在装有约0.2wt%的单分散p(St-NIPAM)水悬液的小瓶中48h,在玻璃基板上沉积得到光子晶体薄膜(PC)。该光子晶体薄膜(光子晶体粒径为668nm)为紫色的光子晶体薄膜,命名为PC668。
c、光子晶体与纳米银构成的复合体的制备
将PC668覆盖在银岛膜上,得到光子晶体薄膜与纳米银薄膜组成的复合体,命名为Ag-PC668。
d、不同基底荧光增强效果比较
用Ag-PC668与纳米银膜(记为Ag)、载玻片(玻璃)、单独的PC668分别做基底,来检测荧光染料AF647分子的荧光强度特性。从图5中a)结果显示,与载玻片相比,AF647在纳米银膜、PC668和Ag-PC668上的荧光强度均显著增加。且在以Ag-PC668为基底时,AF647的荧光强度最强,见图5中a)所示。在减去空白背景后,AF647分子荧光强度直方图见图5中b)所示,其展示了不同基底荧光增强的倍数。结果显示,以Ag-PC668为基底时,荧光增强的倍数达到最高。因此,在定量检测外泌体的浓度时,可以采用Ag-PC668为基底,与金纳米棒共同组成“夹心法”免疫荧光检测,其效果更佳。
5.载玻片上等离子体金膜的制备
将载玻片浸入浓度为3mM的HAuCl4溶液中。每毫升总体积加入20μL氢氧化铵(NH4OH),振荡1min。用去离子水冲洗后,将载玻片浸入浓度为1mM的硼氢化钠(NaBH4)溶液中1min。再次用去离子水冲洗载玻片,将底物浸入1:1的HAuCl4和NH2OH水溶液中(750μM),室温,震荡15-20min。然后用去离子水清洗载玻片,清洗后干燥,并保存在密封容器中,待用。
应当理解的是,本发明的应用不限于上述的举例,对本领域普通技术人员来说,可以根据上述说明加以改进或变换,所有这些改进和变换都应属于本发明所附权利要求的保护范围。
Claims (6)
1.一种基于免疫荧光增强的外泌体定量检测方法,其特征在于,包括步骤:
采用离心法提取外泌体或将待测体液稀释;
将金属纳米粒子荧光探针与外泌体或稀释后的待测体液结合,然后置于金属纳米膜上进行荧光成像,实现外泌体或待测体液中外泌体定量检测。
2.根据权利要求1所述的基于免疫荧光增强的外泌体定量检测方法,其特征在于,所述金属纳米粒子荧光探针由金属纳米棒、外泌体表面标志蛋白抗体和荧光剂结合而成。
3.根据权利要求2所述的基于免疫荧光增强的外泌体定量检测方法,其特征在于,所述金属纳米棒为金纳米棒。
4.根据权利要求2所述的基于免疫荧光增强的外泌体定量检测方法,其特征在于,所述外泌体表面标志蛋白抗体选自抗ALIX抗体、抗Annexin抗体、抗ANXA5抗体、抗CD171抗体、抗CD31抗体、抗CD44抗体、抗CD63抗体、抗CD81抗体、抗CD9抗体、抗Claudins抗体、抗EpCAM抗体、抗FLOT1抗体、抗GM130抗体、抗HLA-G抗体、抗HSP5抗体、抗HSP70抗体、抗HSP90抗体、抗ICAM-1抗体、抗Integrins抗体、抗RAB5抗体、抗SNAP抗体、抗TIM抗体和抗TSG101抗体中的一种。
5.根据权利要求2所述的基于免疫荧光增强的外泌体定量检测方法,其特征在于,所述荧光剂为AF647染料。
6.根据权利要求1所述的基于免疫荧光增强的外泌体定量检测方法,其特征在于,所述金属纳米膜为纳米银膜;
或者,所述金属纳米膜为纳米银膜和形成于所述纳米银膜上的PC668光子晶体膜构成的复合膜;
或者,所述金属纳米膜为纳米金膜。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110648316.1A CN113552345B (zh) | 2021-06-10 | 2021-06-10 | 一种基于免疫荧光增强的外泌体定量检测方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110648316.1A CN113552345B (zh) | 2021-06-10 | 2021-06-10 | 一种基于免疫荧光增强的外泌体定量检测方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113552345A true CN113552345A (zh) | 2021-10-26 |
CN113552345B CN113552345B (zh) | 2024-02-09 |
Family
ID=78130529
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110648316.1A Active CN113552345B (zh) | 2021-06-10 | 2021-06-10 | 一种基于免疫荧光增强的外泌体定量检测方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113552345B (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113933281A (zh) * | 2021-12-14 | 2022-01-14 | 中国农业大学 | 一种基于光纤倏逝波荧光生物传感器的外泌体检测方法 |
Citations (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101339135A (zh) * | 2008-08-18 | 2009-01-07 | 中国科学院化学研究所 | 利用光子晶体提高生物检测灵敏度的方法 |
US20100035335A1 (en) * | 2008-08-08 | 2010-02-11 | Lakowicz Joseph R | Metal-enhanced fluorescence for the label-free detection of interacting biomolecules |
WO2010074083A1 (ja) * | 2008-12-24 | 2010-07-01 | コニカミノルタホールディングス株式会社 | 表面プラズモンおよび蛍光共鳴エネルギー転移を利用した免疫アッセイ |
CN102072891A (zh) * | 2009-11-20 | 2011-05-25 | 中国科学院化学研究所 | 金属修饰的光子晶体生物检测薄膜及其制备方法和用途 |
US20130172207A1 (en) * | 2011-12-28 | 2013-07-04 | The Board Of Trustees Of The Leland Stanford Junior University | Fluorescence enhancing plasmonic nanoscopic gold films and assays based thereon |
CN105352921A (zh) * | 2015-10-13 | 2016-02-24 | 北京科技大学 | 一种基于光子晶体增强荧光的汞离子传感器的制备及应用 |
CN106841613A (zh) * | 2017-01-18 | 2017-06-13 | 上海良润生物医药科技有限公司 | 一种检测外泌体的方法及体系 |
WO2018054390A1 (zh) * | 2016-09-20 | 2018-03-29 | 江南大学 | 一种用于胞内癌症标志物双重检测的卫星状纳米组装体的制备方法及应用 |
CN111157504A (zh) * | 2020-01-14 | 2020-05-15 | 东南大学 | 一种基于光子晶体的外泌体多元检测方法 |
CN111579771A (zh) * | 2020-06-10 | 2020-08-25 | 中国石油大学(华东) | 一种双效荧光增强的免疫分析方法 |
-
2021
- 2021-06-10 CN CN202110648316.1A patent/CN113552345B/zh active Active
Patent Citations (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100035335A1 (en) * | 2008-08-08 | 2010-02-11 | Lakowicz Joseph R | Metal-enhanced fluorescence for the label-free detection of interacting biomolecules |
CN101339135A (zh) * | 2008-08-18 | 2009-01-07 | 中国科学院化学研究所 | 利用光子晶体提高生物检测灵敏度的方法 |
WO2010074083A1 (ja) * | 2008-12-24 | 2010-07-01 | コニカミノルタホールディングス株式会社 | 表面プラズモンおよび蛍光共鳴エネルギー転移を利用した免疫アッセイ |
CN102072891A (zh) * | 2009-11-20 | 2011-05-25 | 中国科学院化学研究所 | 金属修饰的光子晶体生物检测薄膜及其制备方法和用途 |
US20130172207A1 (en) * | 2011-12-28 | 2013-07-04 | The Board Of Trustees Of The Leland Stanford Junior University | Fluorescence enhancing plasmonic nanoscopic gold films and assays based thereon |
CN105352921A (zh) * | 2015-10-13 | 2016-02-24 | 北京科技大学 | 一种基于光子晶体增强荧光的汞离子传感器的制备及应用 |
WO2018054390A1 (zh) * | 2016-09-20 | 2018-03-29 | 江南大学 | 一种用于胞内癌症标志物双重检测的卫星状纳米组装体的制备方法及应用 |
CN106841613A (zh) * | 2017-01-18 | 2017-06-13 | 上海良润生物医药科技有限公司 | 一种检测外泌体的方法及体系 |
CN111157504A (zh) * | 2020-01-14 | 2020-05-15 | 东南大学 | 一种基于光子晶体的外泌体多元检测方法 |
CN111579771A (zh) * | 2020-06-10 | 2020-08-25 | 中国石油大学(华东) | 一种双效荧光增强的免疫分析方法 |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113933281A (zh) * | 2021-12-14 | 2022-01-14 | 中国农业大学 | 一种基于光纤倏逝波荧光生物传感器的外泌体检测方法 |
CN113933281B (zh) * | 2021-12-14 | 2022-03-18 | 中国农业大学 | 一种基于光纤倏逝波荧光生物传感器的外泌体检测方法 |
Also Published As
Publication number | Publication date |
---|---|
CN113552345B (zh) | 2024-02-09 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Luan et al. | Ultrabright fluorescent nanoscale labels for the femtomolar detection of analytes with standard bioassays | |
Atar et al. | A novel QCM immunosensor development based on gold nanoparticles functionalized sulfur-doped graphene quantum dot and h-ZnS-CdS NC for Interleukin-6 detection | |
CN108562569B (zh) | 一种基于表面增强拉曼光谱探针的循环肿瘤细胞检测方法 | |
Liang et al. | Magnetic Fe3O4@ Au composite-enhanced surface plasmon resonance for ultrasensitive detection of magnetic nanoparticle-enriched α-fetoprotein | |
Er et al. | Metal nanoparticles/MoS2 surface-enhanced Raman scattering-based sandwich immunoassay for α-fetoprotein detection | |
TWI418785B (zh) | 將金屬奈米粒子改質以改善表面增進拉曼光譜術(sers)的分析物偵測效果 | |
Zhang et al. | Novel nitrocellulose membrane substrate for efficient analysis of circulating tumor cells coupled with surface-enhanced Raman scattering imaging | |
Zhu et al. | Hydrophobic plasmonic nanoacorn array for a label-free and uniform SERS-based biomolecular assay | |
JP5358648B2 (ja) | 免疫学的測定用青色金ナノ粒子、その製造方法およびそれを用いた測定方法 | |
JP6526810B2 (ja) | 樹脂−白金複合体及びその利用 | |
JP6945445B2 (ja) | プラズモン特異的結合パートナーアッセイにおけるシグナル増幅 | |
EP2482073B1 (en) | Assay method and kit for assay employing sensor chip for fluorescent measuring apparatus utilizing surface plasmon-field enhanced fluorescence spectrometry | |
Pai et al. | Development of a simplified approach for the fabrication of localised surface plasmon resonance sensors based on gold nanorods functionalized using mixed polyethylene glycol layers | |
Oh et al. | Development of a cuvette-based LSPR sensor chip using a plasmonically active transparent strip | |
Lu et al. | Detection of squamous cell carcinoma antigen in cervical cancer by surface-enhanced Raman scattering-based immunoassay | |
Yang et al. | Biotin-streptavidin sandwich integrated PDA-ZnO@ Au nanocomposite based SPR sensor for hIgG detection | |
Emami et al. | Comparison of gold nanoparticle conjugated secondary antibody with non-gold secondary antibody in an ELISA kit model | |
Zhou et al. | Hypersensitive detection of IL-6 on SERS substrate calibrated by dual model | |
KR102257511B1 (ko) | 자성-광학 복합 나노구조체 | |
WO2017046179A1 (en) | End-cap suitable for optical fiber devices and nanoplasmonic sensors | |
JP5196749B2 (ja) | 標的物質検出材料、及びその製造方法 | |
CN113552345B (zh) | 一种基于免疫荧光增强的外泌体定量检测方法 | |
JP6987939B2 (ja) | 金被覆銀ナノプレートの懸濁液並びにその用途及び製造方法 | |
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 | |
Yuan et al. | Plasmon-enhanced fluorescence imaging with silicon-based silver chips for protein and nucleic acid assay |
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 |