CN113376372A - Immunofluorescence colloidal gold nano microsphere material - Google Patents
Immunofluorescence colloidal gold nano microsphere material Download PDFInfo
- Publication number
- CN113376372A CN113376372A CN202110664538.2A CN202110664538A CN113376372A CN 113376372 A CN113376372 A CN 113376372A CN 202110664538 A CN202110664538 A CN 202110664538A CN 113376372 A CN113376372 A CN 113376372A
- Authority
- CN
- China
- Prior art keywords
- colloidal gold
- immunofluorescence
- microsphere
- nano
- nano microsphere
- 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.)
- Pending
Links
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 98
- 239000004005 microsphere Substances 0.000 title claims abstract description 77
- 238000010166 immunofluorescence Methods 0.000 title claims abstract description 28
- 239000000463 material Substances 0.000 title claims abstract description 27
- 239000002096 quantum dot Substances 0.000 claims abstract description 33
- 238000005859 coupling reaction Methods 0.000 claims abstract description 11
- 230000008878 coupling Effects 0.000 claims abstract description 10
- 238000010168 coupling process Methods 0.000 claims abstract description 10
- 125000000524 functional group Chemical group 0.000 claims abstract description 9
- 239000000126 substance Substances 0.000 claims abstract description 8
- 239000002077 nanosphere Substances 0.000 claims description 23
- 238000012986 modification Methods 0.000 claims description 22
- 230000004048 modification Effects 0.000 claims description 22
- 238000000034 method Methods 0.000 claims description 18
- 238000002360 preparation method Methods 0.000 claims description 8
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 7
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 4
- 229910052710 silicon Inorganic materials 0.000 claims description 4
- 239000010703 silicon Substances 0.000 claims description 4
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 claims description 3
- 125000003172 aldehyde group Chemical group 0.000 claims description 3
- 125000004218 chloromethyl group Chemical group [H]C([H])(Cl)* 0.000 claims description 3
- 229910000077 silane Inorganic materials 0.000 claims description 3
- 125000003700 epoxy group Chemical group 0.000 claims description 2
- 238000007306 functionalization reaction Methods 0.000 claims description 2
- -1 sulfonic group Chemical group 0.000 claims description 2
- 125000003396 thiol group Chemical group [H]S* 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 20
- 238000004587 chromatography analysis Methods 0.000 abstract description 14
- 238000000338 in vitro Methods 0.000 abstract description 12
- 238000003745 diagnosis Methods 0.000 abstract description 8
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 239000007790 solid phase Substances 0.000 abstract description 5
- 239000002105 nanoparticle Substances 0.000 abstract description 4
- 108090000623 proteins and genes Proteins 0.000 abstract description 4
- 102000004169 proteins and genes Human genes 0.000 abstract description 4
- 102000039446 nucleic acids Human genes 0.000 abstract description 3
- 108020004707 nucleic acids Proteins 0.000 abstract description 3
- 150000007523 nucleic acids Chemical class 0.000 abstract description 3
- 230000003287 optical effect Effects 0.000 abstract description 3
- 238000011161 development Methods 0.000 abstract description 2
- 230000009977 dual effect Effects 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 53
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 31
- 238000006243 chemical reaction Methods 0.000 description 16
- 238000002156 mixing Methods 0.000 description 15
- 239000000047 product Substances 0.000 description 15
- 239000006228 supernatant Substances 0.000 description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 108010048233 Procalcitonin Proteins 0.000 description 11
- 238000004140 cleaning Methods 0.000 description 11
- CWCXERYKLSEGEZ-KDKHKZEGSA-N procalcitonin Chemical compound C([C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)NCC(O)=O)[C@@H](C)O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCSC)NC(=O)[C@H]1NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)CNC(=O)[C@@H](N)CSSC1)[C@@H](C)O)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 CWCXERYKLSEGEZ-KDKHKZEGSA-N 0.000 description 11
- 238000012360 testing method Methods 0.000 description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 10
- 230000008569 process Effects 0.000 description 10
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 9
- 150000001875 compounds Chemical class 0.000 description 9
- 238000001132 ultrasonic dispersion Methods 0.000 description 9
- 239000012528 membrane Substances 0.000 description 8
- 239000002904 solvent Substances 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 229910052757 nitrogen Inorganic materials 0.000 description 6
- 229920000642 polymer Polymers 0.000 description 6
- 238000005507 spraying Methods 0.000 description 6
- 229920002873 Polyethylenimine Polymers 0.000 description 5
- 229920001213 Polysorbate 20 Polymers 0.000 description 5
- 238000010438 heat treatment Methods 0.000 description 5
- 239000002086 nanomaterial Substances 0.000 description 5
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 5
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 description 4
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 4
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 229920000089 Cyclic olefin copolymer Polymers 0.000 description 4
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 239000002131 composite material Substances 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000004372 Polyvinyl alcohol Substances 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- 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 3
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 239000012876 carrier material Substances 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 235000019441 ethanol Nutrition 0.000 description 3
- 239000010931 gold Substances 0.000 description 3
- 229910052737 gold Inorganic materials 0.000 description 3
- 239000010410 layer Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000010355 oscillation Effects 0.000 description 3
- 229920002451 polyvinyl alcohol Polymers 0.000 description 3
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 3
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 3
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 3
- 239000003761 preservation solution Substances 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- YYROPELSRYBVMQ-UHFFFAOYSA-N 4-toluenesulfonyl chloride Chemical compound CC1=CC=C(S(Cl)(=O)=O)C=C1 YYROPELSRYBVMQ-UHFFFAOYSA-N 0.000 description 2
- 229920002126 Acrylic acid copolymer Polymers 0.000 description 2
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 2
- 102000055006 Calcitonin Human genes 0.000 description 2
- 108060001064 Calcitonin Proteins 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- RVGRUAULSDPKGF-UHFFFAOYSA-N Poloxamer Chemical compound C1CO1.CC1CO1 RVGRUAULSDPKGF-UHFFFAOYSA-N 0.000 description 2
- 229920002845 Poly(methacrylic acid) Polymers 0.000 description 2
- 229920002125 Sokalan® Polymers 0.000 description 2
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Natural products C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 2
- 239000002250 absorbent Substances 0.000 description 2
- 230000002745 absorbent Effects 0.000 description 2
- 239000011149 active material Substances 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- BBBFJLBPOGFECG-VJVYQDLKSA-N calcitonin Chemical compound N([C@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(N)=O)C(C)C)C(=O)[C@@H]1CSSC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1 BBBFJLBPOGFECG-VJVYQDLKSA-N 0.000 description 2
- 229960004015 calcitonin Drugs 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000012412 chemical coupling Methods 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 229920001577 copolymer Polymers 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 description 2
- 239000003365 glass fiber Substances 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 238000010030 laminating Methods 0.000 description 2
- FPYJFEHAWHCUMM-UHFFFAOYSA-N maleic anhydride Chemical compound O=C1OC(=O)C=C1 FPYJFEHAWHCUMM-UHFFFAOYSA-N 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 229960000502 poloxamer Drugs 0.000 description 2
- 229920001983 poloxamer Polymers 0.000 description 2
- 239000004584 polyacrylic acid Substances 0.000 description 2
- 229920000728 polyester Polymers 0.000 description 2
- 239000008213 purified water Substances 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- 235000012239 silicon dioxide Nutrition 0.000 description 2
- RPENMORRBUTCPR-UHFFFAOYSA-M sodium;1-hydroxy-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].ON1C(=O)CC(S([O-])(=O)=O)C1=O RPENMORRBUTCPR-UHFFFAOYSA-M 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 150000003573 thiols Chemical class 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- UUEWCQRISZBELL-UHFFFAOYSA-N 3-trimethoxysilylpropane-1-thiol Chemical compound CO[Si](OC)(OC)CCCS UUEWCQRISZBELL-UHFFFAOYSA-N 0.000 description 1
- 238000009007 Diagnostic Kit Methods 0.000 description 1
- 239000004593 Epoxy Substances 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241001363516 Plusia festucae Species 0.000 description 1
- 238000012356 Product development Methods 0.000 description 1
- 239000006087 Silane Coupling Agent Substances 0.000 description 1
- 125000003277 amino group Chemical group 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
- GHQPBDDZGPAVJP-UHFFFAOYSA-N azanium;methanol;hydroxide Chemical compound N.O.OC GHQPBDDZGPAVJP-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 238000011503 in vivo imaging Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- XTUSEBKMEQERQV-UHFFFAOYSA-N propan-2-ol;hydrate Chemical compound O.CC(C)O XTUSEBKMEQERQV-UHFFFAOYSA-N 0.000 description 1
- 239000011241 protective layer Substances 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- RMAQACBXLXPBSY-UHFFFAOYSA-N silicic acid Chemical compound O[Si](O)(O)O RMAQACBXLXPBSY-UHFFFAOYSA-N 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 125000002088 tosyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1C([H])([H])[H])S(*)(=O)=O 0.000 description 1
- 238000012795 verification 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
- G01N33/543—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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- 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
- G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Nanotechnology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Materials Engineering (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention discloses an immunofluorescence colloidal gold nano microsphere material, relates to the field of in vitro diagnosis, and aims to solve the problems that in the prior art, the fluorescence quantitative detection result is difficult to interpret and high in cost, and the lateral chromatography detection sensitivity with colloidal gold as a carrier is not high; the adopted technical scheme is that two-in-one microspheres obtained by coupling colloidal gold nanoparticles and fluorescent quantum dot nanoparticles through chemical bonds are used as a main solid phase carrier for lateral chromatography, and can be coupled with proteins, nucleic acids and the like through functional groups on the surface of the carrier, so that the carrier has the color development characteristic of colloidal gold, and can realize the characteristics of simple lateral chromatography operation and easy interpretation of results; the quantum dot optical characteristics can realize quantitative detection, the sensitivity is low, and the material and labor cost are reduced under the condition of realizing dual functions.
Description
Technical Field
The invention relates to the field of in-vitro diagnosis, in particular to an immunofluorescence colloidal gold nano microsphere material with the coexistence of qualitative and quantitative properties.
Background
In Vitro diagnosis, i.e., ivd (in Vitro diagnosis), refers to products and services for obtaining clinical diagnosis information by detecting human body samples (blood, body fluid, tissue, etc.) outside the human body, and further determining diseases or body functions.
The in vitro diagnostic industry and the laboratory medicine constitute an organic whole which is not only distinguished but also closely related to each other. The in vitro diagnostics industry is the "tools" and "weapons" of laboratory medicine, while laboratory medicine is the "users" and "markets" of the in vitro diagnostics industry, both of which share the common objective of performing in vitro diagnostics. About 80% of clinical diagnostic information comes from in vitro diagnosis, and the cost thereof accounts for less than 20% of medical costs. In vitro diagnosis has become an increasingly important component in the prevention, diagnosis and treatment of human diseases.
The nano polymeric microspheres are various in types and are used in different fields; in the field of IVD, the lateral chromatography technology has been developed for many years, qualitative detection represented by colloidal gold cannot meet the requirements of people, and quantitative detection gradually begins to develop. And in the lateral chromatography direction, fluorescent microspheres are used as carrier materials for quantitative detection.
Fluorescent microspheres are a special class of functional microspheres, which generally have diameters of nanometer to micrometer, and are coated with fluorescent substances on the surface or loaded with fluorescent substances in the structure, and can emit fluorescence under excitation light. Due to the characteristics of stable structure, stable luminous efficiency and the like, the fluorescent microspheres are widely applied to the fields of marking, tracing, detecting, in-vivo imaging, immune medicine, high-flux drug screening and the like.
In the IVD field, the colloidal gold test paper strip has the characteristics of easy interpretation of results and low detection cost, so that colloidal gold can enter daily family life, but the lateral chromatography of fluorescence quantitative detection cannot meet the requirement, and the cost is greatly increased due to the need of matched quantitative detection equipment.
In the lateral chromatography detection using colloidal gold as a carrier, quantitative and sensitivity problems always disturb the developers of general diagnostic kits. But the solid phase carrier material which can realize qualitative simple and easy interpretation, can assist in improving the sensitivity and realize quantification is not realized in the market.
At present, in order to realize qualitative and quantitative functions, some manufacturers usually mix a colloidal gold marker with a fluorescent marker, and although the qualitative and quantitative functions can be realized, the investment of active materials is doubled, and meanwhile, the cost of labor and the like is multiplied due to the marking and the use of the two materials.
Disclosure of Invention
In view of the problems in the prior art, the invention discloses an immunofluorescence colloidal gold nano-microsphere material, which adopts the technical scheme that the immunofluorescence colloidal gold nano-microsphere material comprises a colloidal gold nano-microsphere, a fluorescence quantum dot nano-microsphere and a nano-microsphere surface polymer modification group, wherein the surface of the colloidal gold nano-microsphere is functionalized and is coupled with the fluorescence quantum dot nano-microsphere through a chemical bond, the diameter can be 10-500nm, the fluorescence emission wavelength range is 400-800nm, and the color development characteristic of the colloidal gold nano-microsphere is still maintained on the basis.
As a preferred technical solution of the present invention, the functional group on the surface of the colloidal gold nanosphere may be carboxyl, amino, aldehyde, epoxy, sulfonic acid, chloromethyl, hydroxyl, etc.; the nano-microsphere can be used in the field of IVD (in-vitro chromatography), can be used as a solid phase carrier material of lateral chromatography, can perform coupling reaction with protein, nucleic acid and the like through functional groups on the surface of the nano-microsphere, can realize qualitative judgment through the performance of colloidal gold in the lateral chromatography, and can also perform quantitative reading through detecting the emitted light of quantum dots.
As a preferred technical scheme of the invention, the fluorescent quantum dot nano microsphere is coupled on the surface of the colloidal gold nano microsphere.
As a preferred technical scheme of the invention, the colloidal gold nanospheres are coupled on the surface of the fluorescent quantum dot nanospheres.
The invention discloses a preparation method of an immunofluorescence colloidal gold nano microsphere material, which adopts the technical scheme that the preparation method comprises the following steps:
step a, performing film covering protection on the surface of the colloidal gold nanosphere;
b, performing functionalized modification on the surface of the colloidal gold nanosphere obtained in the step a;
c, coupling the colloidal gold nano-microspheres obtained in the step b with fluorescent quantum dot nano-microspheres;
and d, performing surface modification on the two-in-one nano microsphere obtained in the step c.
As a preferred embodiment of the present invention, the protection of the coating film in step a may be, but is not limited to, a silicon-clad shell treatment; using monomer polymer to react with nano-microspheres such as colloidal gold and the like, and then covering the nano-microspheres such as colloidal gold and the like with a protective layer; the nano materials such as the colloidal gold are protected, and meanwhile, the colloidal gold is endowed with a stable structure, and the spherical shape and the structure of the materials are protected.
In a specific implementation process, the following steps are taken:
step 1, taking a high-concentration colloidal gold solution (more than or equal to 1OD), and adding a solution with a mass-to-volume ratio of 1:1, the high molecular materials can be PVP (polyvinylpyrrolidone), PVA (polyvinyl alcohol), poloxamer and the like; uniformly mixing colloidal gold and a high polymer material, and standing for reaction to obtain a solution A; repeated verification confirms that the higher the concentration of the colloidal gold is, the higher the film attaching efficiency of the polymer is;
step 2, adding ammonia water-ethanol or ammonia water-methanol, ammonia water-isopropanol and other solutions with the volume ratio of 1:19 into the solution A obtained in the step 1, and stirring or shaking and uniformly mixing to obtain a solution B;
step 3, adding a silicic acid compound-containing solution into the solution B obtained in the step 2, wherein the solvent is consistent with that of the solution B and is correspondingly methanol, ethanol or isopropanol and the like; further, the volume ratio of silicic acid to solvent is generally 1: 9; obtaining a solution C;
step 4, centrifuging the solution C obtained in the step 3 to remove supernatant, repeatedly washing the solution C for 4-5 times by adopting 10000g of centrifugal force and preferably selecting a corresponding solvent, and redissolving and dispersing to obtain the colloidal gold nanomaterial with silica shells attached to the surface; when cleaning, firstly centrifuging and removing supernatant, then redissolving with solvent, and centrifuging again and removing supernatant and redissolving, which is a complete one-time cleaning process.
In a preferred embodiment of the present invention, the functional modification in step b may be, but is not limited to, a thiol modification.
In the specific implementation process, the following steps are adopted:
step 1, reacting the colloidal gold nanospheres with the silicon shells attached to the surfaces obtained in the step a with a silane coupling agent solution containing mercapto groups, wherein the solvent is 1:19, correspondingly, the solvent is consistent with the surface silica shell colloidal gold nano material to obtain a solution D;
and 2, centrifuging the solution D obtained in the step 1 under 10000g, after removing a supernatant, preferably redissolving by a DMSO (dimethyl sulfoxide) or DMF (dimethyl formamide) solution, washing for 3-4 times under the same condition, and after the last redissolution, performing ultrasonic dispersion and suspension to obtain a colloidal gold solution E with the surface modified sulfydryl.
As a preferred embodiment of the present invention, the coupling mode in step c may be, but is not limited to, silane coupling.
In the specific implementation process, reacting the fluorescent quantum dot solution with the colloidal gold solution E obtained in the step b to obtain the colloidal gold-fluorescent quantum dot coupled microsphere, wherein the fluorescent quantum dot is dissolved in DMSO or DMF, further kept consistent with the solvent of the solution E, centrifuged under 10000g, repeatedly washed for 3-4 times, and finally redissolved to obtain a solution F.
As a preferred embodiment of the present invention, in the step d, the surface modification of the two-in-one nanoparticle may be, but not limited to, the modification of carboxyl, amino, aldehyde, sulfonic acid, and the like.
In the specific implementation process, the following steps are adopted:
step 1, adding a polymer monomer solution into the solution F obtained in the step c, wherein the polymer can be polyethyleneimine, polyacrylic acid, polymethacrylic acid, acrylic acid and styrene copolymer, acrylic acid and olefin copolymer, maleic anhydride and olefin copolymer and the like, and reacting at the temperature of 30-90 ℃. Obtaining a colloidal gold fluorescent quantum dot two-in-one microsphere mixed solution G with the surface modified with functional groups;
and 2, centrifuging the mixed solution G obtained in the step 1 under the condition of 10000G, removing supernatant, cleaning for 3-4 times by using 0.01M sodium hydroxide solution, further cleaning for 3-4 times by using purified water, and finally performing ultrasonic dispersion to obtain the surface-modified colloidal gold fluorescent quantum dot two-in-one nano microsphere.
The invention has the beneficial effects that: according to the invention, the colloidal gold nanospheres and the fluorescent quantum dot nanospheres are coupled through chemical bonds to obtain two-in-one microspheres which are used as main solid phase carriers for lateral chromatography and can be coupled with proteins, nucleic acids and the like through functional groups on the surfaces of the microspheres, so that the colloidal gold developing characteristic is achieved, and the characteristics of simple lateral chromatography operation and easily-interpreted result can be realized; the quantum dot optical characteristics are provided, quantitative detection can be realized, the problem of low sensitivity of qualitative products is solved, and meanwhile, the material saving and labor cost are reduced under the condition of realizing double functions.
Furthermore, for research and development manufacturers, more selection schemes are provided for developed products, and for the same developed product, the same product can be selected to be popularized as a qualitative product and also can be selected to be popularized as a quantitative product, namely, two products are developed only once, so that the product development period and the product investment are reduced.
Further, for the user, the low value region is also referred to as "gray region", and the simple colloidal gold method cannot accurately determine the negative or positive by naked eyes, but can do so by optical property detection, thereby reducing the probability of missed detection.
Furthermore, the colloidal gold labeled antibody is a physical adsorption process, generally, the labeling efficiency of the physical adsorption labeled antibody is low and unstable, and the colloidal gold fluorescent quantum dot microspheres are surface modification functional groups, can label the antibody through chemical coupling, are high in labeling efficiency and stable, and improve the product quality.
Furthermore, the use of active materials can be greatly reduced under the condition of meeting the requirements of qualitative and quantitative properties, the reaction performance can be improved, and the investment of quantitative product cost can be reduced without increasing the investment of instruments and equipment while meeting the requirements of quantitative properties.
Drawings
FIG. 1 is a schematic diagram of a structure of a colloidal gold fluorescent quantum dot nanomaterial.
FIG. 2 is a flow chart of the preparation method of the immunofluorescence colloidal gold nano microsphere material.
Detailed Description
Example 1
As shown in figures 1 and 2, the invention discloses an immunofluorescence colloidal gold nano-microsphere material, which adopts the technical scheme that the immunofluorescence colloidal gold nano-microsphere material comprises a colloidal gold nano-microsphere, a fluorescence quantum dot nano-microsphere and a nano-microsphere surface polymer modification group, wherein the surface of the colloidal gold nano-microsphere is functionalized and is coupled with the fluorescence quantum dot nano-microsphere through a chemical bond.
As a preferred technical solution of the present invention, the functional group on the surface of the colloidal gold nanoparticle microsphere may be a carboxyl group, an amino group, an aldehyde group, an epoxy group, a sulfonic group, a chloromethyl group, or a hydroxyl group.
As a preferred technical scheme of the invention, the fluorescent quantum dot nano microsphere is coupled on the surface of the colloidal gold nano microsphere.
The invention discloses a preparation method of an immunofluorescence colloidal gold nano microsphere material, which adopts the technical scheme that the preparation method comprises the following steps:
step a, performing film covering protection on the surface of the colloidal gold nanosphere;
b, performing functionalized modification on the surface of the colloidal gold nanosphere obtained in the step a;
c, coupling the colloidal gold nano-microspheres obtained in the step b with fluorescent quantum dot nano-microspheres;
and d, performing surface modification on the two-in-one nano microsphere obtained in the step c.
As a preferred technical solution of the present invention, the coating protection in step a adopts a silicon-coated shell treatment, specifically adopting the following steps:
step 1, taking 500 mu L of 200OD 40nm colloidal gold solution, adding 100 mu L of PVP aqueous solution with the concentration of 50%, uniformly mixing, and standing for reacting for 15 minutes;
step 2, adding 100 mu L of ethanol solution containing 5% TEOS, mixing uniformly, and carrying out oscillation reaction for 0.5 hour;
step 3, centrifugal cleaning is carried out for 4 times by using absolute ethyl alcohol (the centrifugal force is 10000g, 10 min/time, and ultrasonic dispersion is adopted in the cleaning re-dissolving process);
and 4, ultrasonically dispersing and dissolving the mixture into 500 mu L of ethanol for later use.
As a preferred technical solution of the present invention, the functionalization modification in the step b is a thiol modification, and specifically comprises the following steps:
step 5, dropwise adding 1% of (3-mercaptopropyl) trimethoxysilane into the silicon-coated colloidal gold by 60 mu L, and stirring for 1 hour at room temperature;
and 6, centrifuging to remove the supernatant, and washing and redissolving the supernatant to 500uL by using a DMSO solution.
As a preferred technical solution of the present invention, the coupling manner in step c is silane coupling, specifically including the following steps:
step 7, dissolving the oil-soluble quantum dots to 5mg/mL by using DMSO, adding 100 mu L of the oil-soluble quantum dots into the colloidal gold with sulfhydrylation surface, and reacting for 0.5 hour at room temperature;
and 8, centrifugally cleaning the product obtained in the step 1, and dispersing the product into 500 mu L of DMSO solution to obtain the colloidal gold and quantum dot composite functional material.
As a preferred technical scheme of the present invention, in the step d, the surface modification of the two-in-one nanosphere adopts the following steps:
step 9, dissolving polyacrylic acid to 10mg/mL by using a DMSO solution, adding the solution into the solution, heating to 90 ℃, and reacting for 4 hours under the protection of nitrogen;
step 10, centrifuging to remove supernatant, and washing with 0.01M sodium hydroxide aqueous solution for 4 times (centrifugal force 10000g, 10 min/time, ultrasonic dispersion is adopted in the washing redissolution process);
and 11, dispersing the precipitate in 500 mu L of purified water to obtain the surface carboxyl functionalized colloidal gold and quantum dot composite microsphere functional material, namely the immunofluorescence colloidal gold nano material.
Example 2
This example differs from example 1 in that: the colloidal gold nanospheres are coupled on the surfaces of the fluorescent quantum dot nanospheres.
Step 1, taking 500 mu L of 200OD 40nm colloidal gold solution, adding 100 mu L of poloxamer aqueous solution with the concentration of 50%, uniformly mixing, and standing for reaction for 15 minutes;
in the step 9, the polymethacrylic acid is dissolved to 10mg/mL by DMSO, added into the solution, heated to 90 ℃, and reacted for 4 hours under the protection of nitrogen.
Example 3
This example differs from example 1 in that: step 2, adding 100 mu L of isopropyl alcohol solution containing 5% TEOS, uniformly mixing, and carrying out oscillation reaction for 0.5 hour;
step 3, centrifugal cleaning is carried out for 4 times by using anhydrous isopropanol (the centrifugal force is 10000g and 10 min/time, and ultrasonic dispersion is adopted in the cleaning redissolution process);
step 4, ultrasonically dispersing the mixture into 500 mu L of isopropanol for later use;
and 9, dissolving the polyethyleneimine into 10mg/mL of DMSO, adding the dissolved polyethyleneimine into the solution, heating to 90 ℃, and reacting for 4 hours under the protection of nitrogen.
Example 4, this example differs from example 1 in that: step 1, taking 500 mu L of 200OD 40nm colloidal gold solution, adding 100 mu L of 50% PVA aqueous solution, uniformly mixing, and standing for reaction for 15 minutes;
and 9, dissolving the polyethyleneimine into 10mg/mL of DMSO, adding the dissolved polyethyleneimine into the solution, heating to 90 ℃, and reacting for 4 hours under the protection of nitrogen.
Example 5
Step 2, adding 100 mul of methanol solution containing 5 percent TEOS, uniformly mixing, and carrying out oscillation reaction for 0.5 hour;
step 3, centrifugal cleaning is carried out for 4 times by using anhydrous methanol (the centrifugal force is 10000g, 10 min/time, and ultrasonic dispersion is adopted in the cleaning redissolution process);
step 4, ultrasonically dispersing the mixture into 500 mu L of methanol for later use;
step 9, dissolving acrylic acid and styrene copolymer, acrylic acid and olefin copolymer, maleic anhydride and olefin copolymer and the like to 10mg/mL by using DMSO, adding the dissolved solution into the solution, heating the solution to 90 ℃, and reacting the solution for 4 hours under the protection of nitrogen
Example 6
This example differs from example 1 in that: and step 9, dissolving the tosyl, the tosyl chloride, the hydroxyl reaction product and the like to 10mg/mL by using DMSO, adding the dissolved solution into the solution, heating the solution to 90 ℃, and reacting the solution for 4 hours under the protection of nitrogen.
Comparative detection test
The preparation method of the immunofluorescence colloidal gold nanosphere procalcitonin test strip comprises the following steps:
1. combining the obtained immunofluorescence colloidal gold nano-microspheres with active proteins such as antibodies or antigens in a chemical coupling mode to obtain an immunofluorescence colloidal gold conjugate; in the present invention, the labeled antibody is a PCT antibody of murine origin;
1.1 adding 15mg of EDC and 50mg of sulfo-NHS into 50ul of immunofluorescence colloidal gold nano-microspheres, vortex mixing uniformly, and reacting for 15min at 37 ℃;
1.2 after the reaction is completed, the supernatant is centrifuged off, and the centrifugation is carried out (centrifugal force 10000g, centrifugation time 8 min).
1.3 Add 1mL of 0.05M TB 8.0 and disperse by sonication.
1.4 adding 20 mu g of procalcitonin monoclonal antibody, placing on a shaking and uniformly mixing device, and reacting for 2h at room temperature;
1.5 then adding 100 mul 10% BSA, blocking the unmarked sites, placing on a shaking and mixing device, and reacting for 30min at room temperature;
1.6 after the reaction is finished, centrifuging 10000g of the liquid for 8min, discarding the supernatant, adding 5mL of a preservation solution (pH8.00.2M TB buffer solution containing 1% BSA, 10% sucrose and 0.5% Tween-20), and performing ultrasonic dispersion to finish marking;
2. using a gold mark three-dimensional gold spot film spraying instrument, setting parameters of 4 mu L/cm, spraying the 1.6 marked compound on polyester fiber DL42, and cutting 7mm by 300mm for later use to obtain a fluorescent nano microsphere marked compound pad; 3. Coating another calcitonin original monoclonal antibody and goat anti-mouse IgG on a nitrocellulose membrane by using a gold-labeled three-dimensional gold-spraying dot membrane instrument according to the parameter of 1 mu L/cm, and drying for 16 hours at 37 ℃ to be used as a reaction membrane;
4. soaking glass fiber in 0.1M PBS solution containing 0.5% Tween-20, drying at 37 deg.C for 16 hr, and cutting into sample pad of 17mm × 300 mm;
5. the sample pad, the fluorescent nano microsphere marked compound pad, the reaction membrane and the absorbent paper (22mm multiplied by 300mm) are adhered and assembled on the PVC bottom plate in a mode of laminating layer by layer, and the obtained product is used as a procalcitonin determination test strip after being cut into strips.
Preparation of the control: the test paper strip for the procalcitonin marked by the fluorescence-colloidal gold comprises the following steps:
1. taking 50ul of 5mg/mL fluorescent quantum dot microspheres with carboxyl on the surface, adding 15mg of EDC and 50mg of sulfo-NHS, uniformly mixing by vortex, and reacting for 15min at 37 ℃; after the reaction is finished, centrifuging to remove the supernatant, and centrifuging under the condition of 10000g for 8 min; adding 1ml of 0.05M TB 8.0, and performing ultrasonic dispersion; adding 2 mu g procalcitonin monoclonal antibody, placing on a shaking mixer, and reacting at room temperature for 2 h; then adding 100 mu L10% BSA, blocking the unmarked sites, placing on a shaking mixer, and reacting for 30min at room temperature; after the reaction is finished, centrifuging the liquid for 8min under the centrifugal force of 10000g, removing supernatant, adding 5mL of preservation solution (pH8.00.2M TB buffer solution containing 1% BSA, 10% sucrose and 0.5% Tween-20), and ultrasonically dispersing; preparing a fluorescence labeling compound;
2. taking 1mL of 1OD colloidal gold solution, adding 20 mu g of procalcitonin monoclonal antibody, placing on a shaking and uniformly mixing device, and reacting for 2h at room temperature; then adding 100 mul 10% BSA, blocking the unmarked sites, placing on a shaking mixer, and reacting for 30min at room temperature; after the reaction is finished, centrifuging the liquid for 8min under the centrifugal force of 10000g, removing the supernatant, adding 5mL of preservation solution (pH8.00.2M TB buffer solution containing 1% BSA, 10% sucrose and 0.5% Tween-20), and performing ultrasonic dispersion; prepared as a fluorescently labeled complex.
3. And (3) mixing the composite microspheres 1 and 2 according to the ratio of 1:1, mixing in proportion to serve as a mixed marking compound of the control test strip;
4. using a gold mark three-dimensional gold spraying point film instrument, setting parameters of 4 mu L/cm, spraying the 3 mixed labeled compound on polyester fiber DL42, and cutting 7mm x 300mm for later use, wherein the labeled compound is called a labeled compound pad.
5. Coating another calcitonin protomonoclonal antibody and goat anti-mouse IgG on the nitrocellulose membrane by a gold-labeled three-dimensional gold-spraying membrane instrument according to the parameter of 1 mu L/cm, and drying for 16 hours at 37 ℃ to be used as a reaction membrane.
6. The glass fiber was soaked in 0.1M PBS containing 0.5% Tween-20, and then dried at 37 ℃ for 16 hours, and cut into 17mm by 300mm as a sample pad.
7. The sample pad, the fluorescent nano microsphere labeled compound pad, the reaction membrane and the absorbent paper (22mm multiplied by 300mm) are adhered and assembled on the PVC bottom plate in a layer-by-layer laminating manner, and the obtained product is cut into strips to be used as a procalcitonin determination test strip.
The quantitative detection result of the immunofluorescence colloidal gold nanosphere procalcitonin is as follows:
the qualitative interpretation result of the immunofluorescence colloidal gold nanosphere procalcitonin is as follows:
the quantitative detection result of the fluorescence-colloidal gold mixed labeled procalcitonin test strip is as follows:
the qualitative detection result of the fluorescence-colloidal gold mixed labeled procalcitonin test strip is as follows:
the following conclusions can be drawn from the above comparative tests:
1. the colloidal gold fluorescent quantum dot two-in-one nano microsphere is used as an in-vitro diagnosis chromatography test strip of a solid phase carrier, and qualitative and quantitative detection can be realized simultaneously.
2. Compared with the pure colloidal gold qualitative property, the two-in-one microsphere has qualitative aspect, sensitivity and linear composite requirements.
3. Compared with the pure colloidal gold qualitative method, the quantitative method has the advantages that the detection sensitivity is greatly improved in the aspect of two-in-one microsphere quantification.
The method couples the colloidal gold and the fluorescent quantum dot microspheres together by a chemical method, performs surface functional group modification on the coupled microspheres, is used in the IVD field and the lateral chromatography, solves the problem of low sensitivity of the colloidal gold, simultaneously retains the characteristic of easy interpretation of the colloidal gold, and can perform quantitative interpretation. Particularly, under the special use condition, such as the 2019-COV new crown detection kit, especially for a household self-inspection user, the low-sensitivity judgment can be carried out only by one ultraviolet flashlight without adding matched equipment, so that the unnecessary cost is reduced.
Although the present invention has been described in detail with reference to the specific embodiments, the present invention is not limited to the above embodiments, and various changes and modifications without inventive changes may be made within the knowledge of those skilled in the art without departing from the spirit of the present invention.
Claims (9)
1. An immunofluorescence colloidal gold nano microsphere material is characterized in that: the nano-microsphere is functionalized on the surface and coupled with the fluorescent quantum dot nano-microsphere through a chemical bond.
2. The immunofluorescence colloidal gold nanosphere material of claim 1, wherein: the functional group on the surface of the immunofluorescence colloidal gold nano microsphere can be carboxyl, amino, aldehyde group, epoxy group, sulfonic group, chloromethyl and hydroxyl.
3. The immunofluorescence colloidal gold nanosphere material of claim 1, wherein: the fluorescent quantum dot nano microsphere is coupled on the surface of the colloidal gold nano microsphere.
4. The immunofluorescence colloidal gold nanosphere material of claim 1, wherein: the colloidal gold nanospheres are coupled on the surfaces of the fluorescent quantum dot nanospheres.
5. A preparation method of an immunofluorescence colloidal gold nano microsphere material is characterized by comprising the following steps:
step a, performing film covering protection on the surface of the colloidal gold nanosphere;
b, performing functionalized modification on the surface of the colloidal gold nanosphere obtained in the step a;
c, coupling the colloidal gold nano-microspheres obtained in the step b with fluorescent quantum dot nano-microspheres;
and d, performing surface modification on the two-in-one nano microsphere obtained in the step c.
6. The method for preparing immunofluorescence colloidal gold nano microsphere material according to claim 5, wherein: and (c) the film covering protection in the step a adopts silicon-coated shell treatment.
7. The method for preparing immunofluorescence colloidal gold nano microsphere material according to claim 5, wherein: the functionalization modification in the step b is a thiol modification.
8. The method for preparing immunofluorescence colloidal gold nano microsphere material according to claim 5, wherein: the coupling mode in the step c adopts silane coupling.
9. The method for preparing immunofluorescence colloidal gold nano microsphere material according to claim 5, wherein: in the step d, the surface modification of the two-in-one nano microsphere adopts the modification of carboxyl, amino, aldehyde group and sulfonic group.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110664538.2A CN113376372A (en) | 2021-06-16 | 2021-06-16 | Immunofluorescence colloidal gold nano microsphere material |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110664538.2A CN113376372A (en) | 2021-06-16 | 2021-06-16 | Immunofluorescence colloidal gold nano microsphere material |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113376372A true CN113376372A (en) | 2021-09-10 |
Family
ID=77574585
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110664538.2A Pending CN113376372A (en) | 2021-06-16 | 2021-06-16 | Immunofluorescence colloidal gold nano microsphere material |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113376372A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115958193A (en) * | 2022-12-15 | 2023-04-14 | 郑州凌思生物科技有限责任公司 | Colloidal gold-PS microsphere composite microsphere and preparation method thereof |
Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102556959A (en) * | 2011-12-30 | 2012-07-11 | 中国科学院苏州纳米技术与纳米仿生研究所 | Preparation method of metal nanoparticle dimer |
| CN103048314A (en) * | 2012-10-25 | 2013-04-17 | 宁波大学 | Electrochemical luminescence immune sensor built by mesoporous material loading quantum dots and coated by nanogold, and detection method of HIV (human immunodeficiency virus) |
| CN103911142A (en) * | 2014-03-26 | 2014-07-09 | 京东方科技集团股份有限公司 | Blue quantum dot composite particles, as well as preparation method, photoelectric element and photoelectric device thereof |
| CN104698171A (en) * | 2013-12-04 | 2015-06-10 | 内蒙古农业大学 | Colloidal gold and light-emitting quantum dot dimer based selectable second-level sensitivity lateral chromatography rapid detection method |
| CN105885843A (en) * | 2016-04-18 | 2016-08-24 | 上海科炎光电技术有限公司 | Up-conversion luminescent material for visual rapid detection |
| CN106645754A (en) * | 2016-12-30 | 2017-05-10 | 中南民族大学 | Photoelectric immune sensor based on water-soluble Cd-Ag-Te quantum dot/nano-gold composite materials |
| CN106680496A (en) * | 2016-12-21 | 2017-05-17 | 武汉大学 | Preparation of colorimetric and fluorescent double-signal nanospheres and application of colorimetric and fluorescent double-signal nanospheres to immunochromatographic quantitative detection |
| CN108414756A (en) * | 2017-10-25 | 2018-08-17 | 深圳市罗湖区人民医院 | Preparation method that is a kind of while detecting the protein chip of four bladder carcinoma markers in urine |
| CN109061205A (en) * | 2018-08-09 | 2018-12-21 | 江西中德生物工程股份有限公司 | Detect the double colour developing qualitative, quantitative immuno-chromatographic test paper strips of fluorescent microsphere-colloidal gold and preparation method thereof of Ractopamine |
| KR102064326B1 (en) * | 2019-07-17 | 2020-01-09 | 경북대학교 산학협력단 | Gold nanoparticle-phosphor hybrid material and its preparing methods |
| CN110776915A (en) * | 2019-08-29 | 2020-02-11 | 浙江工业大学 | Fluorescent/colorimetric bifunctional microsphere based on multi-level assembly structure and preparation method thereof |
| CN111024943A (en) * | 2019-12-20 | 2020-04-17 | 吉林农业大学 | Switch-on/off type composite fluorescent nano probe for rapid detection of salmonella and preparation method thereof |
| CN112251232A (en) * | 2020-10-22 | 2021-01-22 | 中国人民解放军军事科学院军事医学研究院 | Difunctional quantum dot microsphere composite nanomaterial, preparation method and application thereof |
| CN112763705A (en) * | 2020-12-11 | 2021-05-07 | 中国科学院长春应用化学研究所 | Method for quantitatively detecting antigen |
-
2021
- 2021-06-16 CN CN202110664538.2A patent/CN113376372A/en active Pending
Patent Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102556959A (en) * | 2011-12-30 | 2012-07-11 | 中国科学院苏州纳米技术与纳米仿生研究所 | Preparation method of metal nanoparticle dimer |
| CN103048314A (en) * | 2012-10-25 | 2013-04-17 | 宁波大学 | Electrochemical luminescence immune sensor built by mesoporous material loading quantum dots and coated by nanogold, and detection method of HIV (human immunodeficiency virus) |
| CN104698171A (en) * | 2013-12-04 | 2015-06-10 | 内蒙古农业大学 | Colloidal gold and light-emitting quantum dot dimer based selectable second-level sensitivity lateral chromatography rapid detection method |
| CN103911142A (en) * | 2014-03-26 | 2014-07-09 | 京东方科技集团股份有限公司 | Blue quantum dot composite particles, as well as preparation method, photoelectric element and photoelectric device thereof |
| CN105885843A (en) * | 2016-04-18 | 2016-08-24 | 上海科炎光电技术有限公司 | Up-conversion luminescent material for visual rapid detection |
| CN106680496A (en) * | 2016-12-21 | 2017-05-17 | 武汉大学 | Preparation of colorimetric and fluorescent double-signal nanospheres and application of colorimetric and fluorescent double-signal nanospheres to immunochromatographic quantitative detection |
| CN106645754A (en) * | 2016-12-30 | 2017-05-10 | 中南民族大学 | Photoelectric immune sensor based on water-soluble Cd-Ag-Te quantum dot/nano-gold composite materials |
| CN108414756A (en) * | 2017-10-25 | 2018-08-17 | 深圳市罗湖区人民医院 | Preparation method that is a kind of while detecting the protein chip of four bladder carcinoma markers in urine |
| CN109061205A (en) * | 2018-08-09 | 2018-12-21 | 江西中德生物工程股份有限公司 | Detect the double colour developing qualitative, quantitative immuno-chromatographic test paper strips of fluorescent microsphere-colloidal gold and preparation method thereof of Ractopamine |
| KR102064326B1 (en) * | 2019-07-17 | 2020-01-09 | 경북대학교 산학협력단 | Gold nanoparticle-phosphor hybrid material and its preparing methods |
| CN110776915A (en) * | 2019-08-29 | 2020-02-11 | 浙江工业大学 | Fluorescent/colorimetric bifunctional microsphere based on multi-level assembly structure and preparation method thereof |
| CN111024943A (en) * | 2019-12-20 | 2020-04-17 | 吉林农业大学 | Switch-on/off type composite fluorescent nano probe for rapid detection of salmonella and preparation method thereof |
| CN112251232A (en) * | 2020-10-22 | 2021-01-22 | 中国人民解放军军事科学院军事医学研究院 | Difunctional quantum dot microsphere composite nanomaterial, preparation method and application thereof |
| CN112763705A (en) * | 2020-12-11 | 2021-05-07 | 中国科学院长春应用化学研究所 | Method for quantitatively detecting antigen |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115958193A (en) * | 2022-12-15 | 2023-04-14 | 郑州凌思生物科技有限责任公司 | Colloidal gold-PS microsphere composite microsphere and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10209249B2 (en) | Labelled silica nanoparticles for immunochromatographic reagent, immunochromatographic test strip using the same, and immunochromomatographic fluorescence-detecting system or radiation-detecting system | |
| CN104655836B (en) | Immunochromatographic test strip, detection method by using immunochromatographic test strip, and application of immunochromatographic test strip | |
| EP1327885B1 (en) | A kit for the simultaneous diagnosis of a plurality of infectious diseases and a method for preparation of it | |
| CN106680496B (en) | A kind of preparation of colorimetric fluorescence dual signal nanosphere and its application in immunochromatography quantitatively detects | |
| WO2007074722A1 (en) | Fluorescent silica nano-particle, fluorescent nano-material, biochip using the material, and assay method | |
| CN110133252A (en) | For detecting kit and detection method and its application of carcinomebryonic antigen | |
| JPH04301570A (en) | Use of peptide pair having very high specific mutual affinityin in-vitro diagnosis field | |
| CN113376372A (en) | Immunofluorescence colloidal gold nano microsphere material | |
| CN114705869B (en) | Photonic crystal biochip and protein detection method thereof | |
| CN106443003A (en) | Fluorescent quenching test paper strip based on aptamer specific recognition and preparation method and application thereof | |
| CN112630429B (en) | Urine helicobacter pylori antibody detection kit and preparation method thereof | |
| Yang et al. | Handy, rapid and multiplex detection of tumor markers based on encoded silica–hydrogel hybrid beads array chip | |
| CN109507422A (en) | Optics micro-fluidic chip based on polymer and multiple layer metal Nanoparticle Modified | |
| CN110954695B (en) | Norovirus GI and GII type quantum dot joint inspection test strip and preparation method and application thereof | |
| CN107255713B (en) | It can be used for diagnosing the kit of human enterovirus infection | |
| KR101368076B1 (en) | Surface-modified fluorescent silica nanoparticles with excellent aqeous dispersibility and preparing method thereof | |
| CN115837255A (en) | Preparation method of colloidal gold-silicon dioxide composite microspheres | |
| JP5541003B2 (en) | Plasmon excitation sensor chip and assay method using the same | |
| WO2010041736A1 (en) | Assay method using surface plasmon | |
| CN108663523A (en) | It can be used for diagnosing the kit of human enterovirus 71 infection | |
| CN112029728A (en) | Fluorescent magnetic nano-composite and preparation method and application thereof | |
| CN113740538A (en) | Method and product for detecting SARS-CoV-2 novel coronavirus IgM/IgG antibody | |
| CN115254047B (en) | Molecularly imprinted and coated polymer, preparation method and application thereof | |
| WO2021169866A1 (en) | Detection particle suitable for multiplex detection of biomolecules, and preparation method therefor and application thereof | |
| JP2002328130A (en) | Testing piece for immunological measurement method |
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 | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20210910 |