CN111693721A - 基于普鲁士蓝纳米酶标记物的酶联免疫吸附实验的制备方法及应用 - Google Patents
基于普鲁士蓝纳米酶标记物的酶联免疫吸附实验的制备方法及应用 Download PDFInfo
- Publication number
- CN111693721A CN111693721A CN202010571694.XA CN202010571694A CN111693721A CN 111693721 A CN111693721 A CN 111693721A CN 202010571694 A CN202010571694 A CN 202010571694A CN 111693721 A CN111693721 A CN 111693721A
- Authority
- CN
- China
- Prior art keywords
- solution
- enzyme
- prussian blue
- ferroferric oxide
- incubating
- 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
- DCYOBGZUOMKFPA-UHFFFAOYSA-N iron(2+);iron(3+);octadecacyanide Chemical compound [Fe+2].[Fe+2].[Fe+2].[Fe+3].[Fe+3].[Fe+3].[Fe+3].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] DCYOBGZUOMKFPA-UHFFFAOYSA-N 0.000 title claims abstract description 28
- 229960003351 prussian blue Drugs 0.000 title claims abstract description 28
- 239000013225 prussian blue Substances 0.000 title claims abstract description 28
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 21
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 21
- 238000002965 ELISA Methods 0.000 title claims abstract description 16
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 229920001690 polydopamine Polymers 0.000 claims abstract description 34
- 239000002131 composite material Substances 0.000 claims abstract description 26
- 239000011258 core-shell material Substances 0.000 claims abstract description 25
- 239000003550 marker Substances 0.000 claims abstract description 13
- 230000003197 catalytic effect Effects 0.000 claims abstract description 9
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 claims abstract description 9
- 238000007885 magnetic separation Methods 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 46
- 108010048233 Procalcitonin Proteins 0.000 claims description 25
- 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 claims description 25
- 238000005406 washing Methods 0.000 claims description 20
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 17
- 239000012498 ultrapure water Substances 0.000 claims description 17
- 239000008055 phosphate buffer solution Substances 0.000 claims description 15
- 239000000427 antigen Substances 0.000 claims description 14
- 102000036639 antigens Human genes 0.000 claims description 14
- 108091007433 antigens Proteins 0.000 claims description 14
- 238000001514 detection method Methods 0.000 claims description 13
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 12
- 239000011259 mixed solution Substances 0.000 claims description 11
- 238000001035 drying Methods 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 10
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 9
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 8
- -1 polytetrafluoroethylene Polymers 0.000 claims description 8
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 7
- 229940098773 bovine serum albumin Drugs 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 230000009871 nonspecific binding Effects 0.000 claims description 7
- 238000003756 stirring Methods 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 6
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 claims description 6
- 229910052742 iron Inorganic materials 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 239000002105 nanoparticle Substances 0.000 claims description 6
- 239000000047 product Substances 0.000 claims description 6
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 4
- 238000002835 absorbance Methods 0.000 claims description 4
- 238000007789 sealing Methods 0.000 claims description 4
- 239000001632 sodium acetate Substances 0.000 claims description 4
- 235000017281 sodium acetate Nutrition 0.000 claims description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims description 3
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 3
- 239000006185 dispersion Substances 0.000 claims description 3
- 229960003638 dopamine Drugs 0.000 claims description 3
- 239000012467 final product Substances 0.000 claims description 3
- 238000000227 grinding Methods 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- 229920001343 polytetrafluoroethylene Polymers 0.000 claims description 3
- 239000004810 polytetrafluoroethylene Substances 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 239000012265 solid product Substances 0.000 claims description 3
- 239000000725 suspension Substances 0.000 claims description 3
- 238000005303 weighing Methods 0.000 claims description 3
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 239000011248 coating agent Substances 0.000 claims description 2
- 238000000576 coating method Methods 0.000 claims description 2
- 239000012089 stop solution Substances 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims 1
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 abstract description 12
- 239000000758 substrate Substances 0.000 abstract description 5
- 238000005516 engineering process Methods 0.000 abstract description 4
- 229910001447 ferric ion Inorganic materials 0.000 abstract description 4
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 abstract description 3
- 230000005389 magnetism Effects 0.000 abstract description 3
- 238000007254 oxidation reaction Methods 0.000 abstract description 3
- VTLYFUHAOXGGBS-UHFFFAOYSA-N Fe3+ Chemical compound [Fe+3] VTLYFUHAOXGGBS-UHFFFAOYSA-N 0.000 abstract description 2
- 230000002378 acidificating effect Effects 0.000 abstract description 2
- 230000036039 immunity Effects 0.000 abstract description 2
- 238000003018 immunoassay Methods 0.000 abstract description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 239000000090 biomarker Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000000852 hydrogen donor Substances 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 230000036046 immunoreaction Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 150000002505 iron Chemical class 0.000 description 1
- UETZVSHORCDDTH-UHFFFAOYSA-N iron(2+);hexacyanide Chemical compound [Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] UETZVSHORCDDTH-UHFFFAOYSA-N 0.000 description 1
- 239000002114 nanocomposite Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 102000013415 peroxidase activity proteins Human genes 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/583—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with non-fluorescent dye 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
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
- G01N2333/585—Calcitonins
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Nanotechnology (AREA)
- Endocrinology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明公开了基于普鲁士蓝纳米酶标记物的酶联免疫吸附实验的制备方法及应用，涉及纳米科学、生物免疫技术、酶免疫测定技术等领域。本发明利用普鲁士蓝优异的催化性能，制备了负载在四氧化三铁@聚多巴胺核壳复合材料上的普鲁士蓝纳米酶标记物。通过利用四氧化三铁球芯的磁性和聚多巴胺优异的生物相容性，实现了生物分子的固定以及磁分离。通过利用聚多巴胺在酸性条件下具有还原三价铁离子的能力，成功在聚多巴胺表面生成普鲁士蓝纳米酶作为标记物。普鲁士蓝纳米酶通过催化过氧化氢的氧化反应，使底物四甲基联苯胺由无色变为蓝色。该标记物可以适用于多种酶联免疫试验的制备，在科研和临床中具有广泛的应用前景。
Description
技术领域
本发明涉及纳米科学、生物免疫技术、酶免疫测定技术等领域，具体涉及一种酶联免疫反应标记物的制备方法及免疫显色应用。
背景技术
生物标志物检测是目前唯一无创的早期预警癌症等疾病的方法，在临床中对肿瘤的普查、诊断及判断预后等具有十分重要的意义。在众多的生物标志物检测方法中，酶联免疫技术由于其检测迅速，方法简单，特异性强和灵敏性高等优点而成为生物分子定量检测的主要分析技术。
酶联免疫吸附试验是利用吸附在固相载体上的抗原或抗体与酶标记抗体发生特异性结合。向其中加入底物溶液后，底物会在酶的作用下使其所含的供氢体由无色的还原型变成有色的氧化型，发生颜色反应。因此，可以根据底物的颜色反应来判断有无对应的免疫反应，颜色反应的深浅与标本中相应抗体或抗原的量呈正比。这种显色反应可通过酶标仪进行定量测定，通过将抗原抗体反应的特异性和酶化学反应的敏感性相结合，使酶联免疫吸附试验成为一种既特异又灵敏的检测方法。鉴于此，发明一种具有催化性能的纳米酶作为标记物用于酶联免疫吸附试验中，具有重要的现实意义。
通过利用四氧化三铁球芯的磁性和聚多巴胺优异的生物相容性，不仅实现了生物分子的固定以及磁分离，而且大大提高了纳米复合物表面抗体的结合量，实现易于标记的制备效果。普鲁士蓝纳米酶被称为“人工酶过氧化物酶”，因为它对还原过氧化氢表现出高特异性和催化性能。与传统的将铁盐和六氰基铁酸盐与不同氧化态的铁原子混合制备普鲁士蓝的方法不同，使用四氧化三铁@聚多巴胺核壳复合材料还原铁氰化物-铁离子混合物可以在复合材料表面得到普鲁士蓝纳米酶用做标记物，进一步实现对显色液的催化显色。通过酶标仪进行定量测定实现对抗原浓度的检测。
本发明利用普鲁士蓝优异的催化性能，制备了负载在四氧化三铁@聚多巴胺核壳复合材料上的普鲁士蓝纳米酶标记物。通过利用四氧化三铁球芯的磁性和聚多巴胺优异的生物相容性，实现了生物分子的固定以及磁分离。通过利用聚多巴胺酸性条件下具有还原三价铁离子的能力，成功在聚多巴胺表面生成普鲁士蓝纳米酶作为标记物。普鲁士蓝纳米酶通过催化过氧化氢的氧化反应，使底物四甲基联苯胺由无色变为蓝色。
发明内容
本发明的目的之一是提出一种利用聚多巴胺还原铁氰化物-铁离子混合物在四氧化三铁@聚多巴胺核壳复合材料表面制备普鲁士蓝纳米酶的方法。
本发明的目的之二是利用普鲁士蓝对过氧化氢的催化作用，实现显色液颜色的变化。
本发明的目的之三是所制备的标记物用于标记抗体构建夹心型酶联免疫吸附试验，既可以对生物标志物进行高效灵敏的检测，还可以实现定量检测。
本发明的技术方案如下：
基于普鲁士蓝纳米酶标记物的酶联免疫吸附实验的制备方法，包括以下步骤：
（1）四氧化三铁纳米粒子的制备：将0.2 g十二烷基硫酸钠、1.5 g醋酸钠和0.5 g三氯化铁加入到15 mL乙二醇中，在室温下搅拌30分钟；然后将该混合溶液转移到聚四氟乙烯高温反应釜中并在200 ℃下加热12小时；将所得产物用超纯水和乙醇分别洗涤三次；最终产物在35 ℃下真空干燥12小时；干燥后研磨并在室温保存；
（2）四氧化三铁@聚多巴胺核壳复合材料的制备：将100 mg四氧化三铁纳米粒子和200mg多巴胺加入到120 mL、pH=8.8 Tris-HCl缓冲液和100 mL异丙醇混合溶液中；搅拌13小时后，得到黑色的悬浊液；通过磁分离收集产物，并将产物用超纯水洗涤五次以除去未反应的物质，然后在35 ℃真空中干燥；干燥后称重并重新分散在5 mL超纯水中，放于4 ℃冰箱冷藏保存；
（3）取50 μL四氧化三铁@聚多巴胺核壳复合材料分散液用5 mL、0.1 mol·L-1、pH 7.4的磷酸盐缓冲溶液稀释，向其中加入2 mL浓度为10 μg·mL-1的降钙素原检测抗体溶液，在4℃下孵化2 h，离心；再加入100 μL、0.1%的牛血清白蛋白溶液封闭非特异性结合位点，在4℃下孵化2 h，离心分离得到固体产物，将其重新分散于5 mL、0.1 mol·L-1、pH 7.4的磷酸盐缓冲溶液中，制得四氧化三铁@聚多巴胺核壳复合材料标记的降钙素原抗体溶液；
（4）将四氧化三铁@聚多巴胺核壳复合材料标记的降钙素原抗体溶液与0.1 ng·mL-1的抗原以体积比为1:1混合，室温下孵化1 h，离心分离，得到与抗原特异性结合的四氧化三铁@聚多巴胺核壳复合材料标记的降钙素原抗体溶液；
（5）向96微孔板中加入100 μL、1 μg·mL-1降钙素原的抗体溶液，在4 ℃下孵育12 h，用磷酸盐缓冲液洗涤微孔板三次；再加入100 μL、0.1%的牛血清白蛋白溶液封闭微孔板上的非特异性结合位点，室温下孵育1 h，再用磷酸盐缓冲液洗板三次；向其中加入特异性结合上抗原的四氧化三铁@聚多巴胺核壳复合材料标记的降钙素原抗体溶液，孵育1 h，用超纯水洗板三次；向微孔中加入200 μL、1 mmol·L-1的三氯化铁和铁***的混合溶液，反应1 min后去除混合铁液，超纯水洗板三次，制得负载普鲁士蓝纳米酶的标记物；
（6）免疫显色过程：向成功负载普鲁士蓝纳米酶标记物的微孔板中加入100 μL、浓度为1.248 mmol·L-1的3,3',5,5'-四甲基联苯胺显色液和100 μL、浓度为6 mmol·L-1的过氧化氢溶液，微孔板中的溶液由无色变为蓝色；显色时间为15 min，显色结束后加入50 μL、浓度为1 mol·L-1的硫酸终止液，微孔板中的溶液由蓝色变为黄色；用酶标仪测量350 nm-900nm溶液的吸光度。
本发明的有益成果
与现有技术相比，本发明具有如下优点：
1）该标记物制备方法利用聚多巴胺还原铁氰化物-铁离子混合物制备出具有优异催化性能的普鲁士蓝纳米酶，制备简单；
2）该方法制备的标记物利用了普鲁士蓝纳米酶对双氧水的催化作用实现显色液颜色的变化，使酶联免疫吸附试验的效率大大提高；
3）该方法制备的标记物利用四氧化三铁@聚多巴胺核壳复合材料负载免疫物质，达到易于标记的制备效果。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。
实施例1
普鲁士蓝纳米酶标记物的制备方法
（1）四氧化三铁纳米粒子的制备：将0.2 g十二烷基硫酸钠、1.5 g醋酸钠和0.5 g三氯化铁加入到15 mL乙二醇中，在室温下搅拌30分钟；然后将该混合溶液转移到聚四氟乙烯高温反应釜中并在200 ℃下加热12小时；将所得产物用超纯水和乙醇分别洗涤三次；最终产物在35 ℃下真空干燥12小时；干燥后研磨并在室温保存；
（2）四氧化三铁@聚多巴胺核壳复合材料的制备：将100 mg四氧化三铁纳米粒子和200mg多巴胺加入到120 mL、pH=8.8 Tris-HCl缓冲液和100 mL异丙醇混合溶液中；搅拌13小时后，得到黑色的悬浊液；通过磁分离收集产物，并将产物用超纯水洗涤五次以除去未反应的物质，然后在35 ℃真空中干燥；干燥后称重并重新分散在5 mL超纯水中，放于4 ℃冰箱冷藏保存；
（3）取50 μL四氧化三铁@聚多巴胺核壳复合材料分散液用5 mL、0.1 mol·L-1、pH 7.4的磷酸盐缓冲溶液稀释，向其中加入2 mL浓度为10 μg·mL-1的降钙素原检测抗体溶液，在4℃下孵化2 h，离心；再加入100 μL、0.1%的牛血清白蛋白溶液封闭非特异性结合位点，在4℃下孵化2 h，离心分离得到固体产物，将其重新分散于5 mL、0.1 mol·L-1、pH 7.4的磷酸盐缓冲溶液中，制得四氧化三铁@聚多巴胺核壳复合材料标记的降钙素原抗体溶液；
（4）将四氧化三铁@聚多巴胺核壳复合材料标记的降钙素原抗体溶液与0.1 ng·mL-1的抗原以体积比为1:1混合，室温下孵化1 h，离心分离，得到与抗原特异性结合的四氧化三铁@聚多巴胺核壳复合材料标记的降钙素原抗体溶液；
（5）向96微孔板中加入100 μL、1 μg·mL-1降钙素原的抗体溶液，在4 ℃下孵育12 h，用磷酸盐缓冲液洗涤微孔板三次；再加入100 μL、0.1%的牛血清白蛋白溶液封闭微孔板上的非特异性结合位点，室温下孵育1 h，再用磷酸盐缓冲液洗板三次；向其中加入特异性结合上抗原的四氧化三铁@聚多巴胺核壳复合材料标记的降钙素原抗体溶液，孵育1 h，用超纯水洗板三次；向微孔中加入200 μL、1 mmol·L-1的三氯化铁和铁***的混合溶液，反应1 min后去除混合铁液，超纯水洗板三次，制得负载普鲁士蓝纳米酶的标记物。
实施例2
夹心型降钙素原酶联免疫吸附试验的构建
（1）包被：向96微孔板中加入100 μL、1 μg·mL-1降钙素原的抗体溶液，4 ℃下孵化12h；用磷酸盐缓冲液洗板三次，甩干；
（2）封闭：用100 μL 0.1%的牛血清白蛋白溶液封闭非特异性结合位点，37 ℃下孵化1h；
（3）加样：甩去封闭液，加入特异性结合上抗原的四氧化三铁@聚多巴胺核壳复合材料标记的降钙素原抗体溶液，37 ℃下孵化1 h；用磷酸盐缓冲液洗板三次，甩干；
（3）纳米酶：向微孔中加入200 μL三氯化铁和铁***的混合溶液，反应1 min后去除铁液，超纯水洗板三次；
（4）显色：每孔先后加入显色剂A、B各100 μL（显色A液：醋酸钠2.72 g，柠檬酸0.3 g，30%双氧水60 μL，蒸馏水加至100 mL。显色B液：乙二胺四乙酸二钠0.04 g，柠檬酸0.19 g，甘油10 mL，取0.03 g四甲基联苯胺溶于0.6 mL二甲基亚砜中，加入少量水中避光搅拌至溶解，定容至100 mL）；
（5）终止：每孔加入50 μL浓度为1 mol·L-1的硫酸终止液；
（6）检测：于TECAN酶标仪上检测450 nm吸收值。
Claims (4)
1.基于普鲁士蓝纳米酶标记物的酶联免疫吸附实验的制备方法，其特征在于，包括以下步骤：
（1）四氧化三铁纳米粒子的制备：将0.2 g十二烷基硫酸钠、1.5 g醋酸钠和0.5 g三氯化铁加入到15 mL乙二醇中，在室温下搅拌30分钟；然后将该混合溶液转移到聚四氟乙烯高温反应釜中并在200 ℃下加热12小时；将所得产物用超纯水和乙醇分别洗涤三次；最终产物在35 ℃下真空干燥12小时；干燥后研磨并在室温保存；
（2）四氧化三铁@聚多巴胺核壳复合材料的制备：将100 mg四氧化三铁纳米粒子和200mg多巴胺加入到120 mL、pH=8.8 Tris-HCl缓冲液和100 mL异丙醇混合溶液中；搅拌13小时后，得到黑色的悬浊液；通过磁分离收集产物，并将产物用超纯水洗涤五次以除去未反应的物质，然后在35 ℃真空中干燥；干燥后称重并重新分散在5 mL超纯水中，放于4 ℃冰箱冷藏保存；
（3）取50 μL四氧化三铁@聚多巴胺核壳复合材料分散液用5 mL、0.1 mol·L-1、pH 7.4的磷酸盐缓冲溶液稀释，向其中加入2 mL浓度为10 μg·mL-1的降钙素原检测抗体溶液，在4℃下孵化2 h，离心；再加入100 μL、0.1%的牛血清白蛋白溶液封闭非特异性结合位点，在4℃下孵化2 h，离心分离得到固体产物，将其重新分散于5 mL、0.1 mol·L-1、pH 7.4的磷酸盐缓冲溶液中，制得四氧化三铁@聚多巴胺核壳复合材料标记的降钙素原抗体溶液；
（4）将四氧化三铁@聚多巴胺核壳复合材料标记的降钙素原抗体溶液与0.1 ng·mL-1的抗原以体积比为1:1混合，室温下孵化1 h，离心分离，得到与抗原特异性结合的四氧化三铁@聚多巴胺核壳复合材料标记的降钙素原抗体溶液；
（5）向96微孔板中加入100 μL、1 μg·mL-1降钙素原的抗体溶液，在4 ℃下孵育12 h，用磷酸盐缓冲液洗涤微孔板三次；再加入100 μL、0.1%的牛血清白蛋白溶液封闭微孔板上的非特异性结合位点，室温下孵育1 h，再用磷酸盐缓冲液洗板三次；向其中加入特异性结合上抗原的四氧化三铁@聚多巴胺核壳复合材料标记的降钙素原抗体溶液，孵育1 h，用超纯水洗板三次；向微孔中加入200 μL、1 mmol·L-1的三氯化铁和铁***的混合溶液，反应1min后去除混合铁液，超纯水洗板三次，制得负载普鲁士蓝纳米酶的标记物；
（6）免疫显色过程：向成功负载普鲁士蓝纳米酶标记物的微孔板中加入100 μL、浓度为1.248 mmol·L-1的3,3',5,5'-四甲基联苯胺显色液和100 μL、浓度为6 mmol·L-1的过氧化氢溶液，微孔板中的溶液由无色变为蓝色；显色时间为15 min，显色结束后加入50 μL、浓度为1 mol·L-1的硫酸终止液，微孔板中的溶液由蓝色变为黄色；用酶标仪测量350 nm-900nm溶液的吸光度。
2.根据权利要求1所述的普鲁士蓝纳米酶标记物的制备方法，其特征在于，所述的标记物用于对显色液进行催化显色。
3.根据权利要求1所述的制备的普鲁士蓝纳米酶标记物作为降钙素原检测的酶联免疫吸附实验的应用。
4.根据权利要求3所述的酶联免疫吸附实验作为降钙素原检测的应用，其特征在于，检测步骤如下：
（1）包被：向96微孔板中加入100 μL、1 μg·mL-1降钙素原的抗体溶液，4 ℃下孵化12h；用磷酸盐缓冲液洗板三次，甩干；
（2）封闭：用100 μL封闭液封闭，37 ℃下孵化1 h；
（3）加样：甩去封闭液，加入特异性结合上抗原的四氧化三铁@聚多巴胺核壳复合材料标记的降钙素原抗体溶液，37 ℃下孵化1 h；用磷酸盐缓冲液洗板三次，甩干；
（3）纳米酶：向微孔中加入200 uL三氯化铁和铁***的混合溶液，反应1 min后去除铁液，超纯水洗板三次；
（4）显色：每孔先后加入显色剂A、B各100 μL；
（5）终止：每孔加入50 μL终止液；
（6）检测：于TECAN酶标仪上检测450 nm吸收值。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010571694.XA CN111693721A (zh) | 2020-06-22 | 2020-06-22 | 基于普鲁士蓝纳米酶标记物的酶联免疫吸附实验的制备方法及应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010571694.XA CN111693721A (zh) | 2020-06-22 | 2020-06-22 | 基于普鲁士蓝纳米酶标记物的酶联免疫吸附实验的制备方法及应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111693721A true CN111693721A (zh) | 2020-09-22 |
Family
ID=72482684
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010571694.XA Pending CN111693721A (zh) | 2020-06-22 | 2020-06-22 | 基于普鲁士蓝纳米酶标记物的酶联免疫吸附实验的制备方法及应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111693721A (zh) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112945878A (zh) * | 2021-02-03 | 2021-06-11 | 安阳市妇幼保健院(安阳市儿童医院) | 间接光度法测定多巴胺的方法 |
| CN113913183A (zh) * | 2021-09-24 | 2022-01-11 | 山东师范大学 | 一种氧化型tmb纳米材料及其在检测谷胱甘肽中的应用 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109254063A (zh) * | 2018-11-12 | 2019-01-22 | 济南大学 | 一种负载型普鲁士蓝电化学生物传感器标记物的制备方法 |
| CN110261600A (zh) * | 2019-07-02 | 2019-09-20 | 济南大学 | 一种基于四氧化三铁/普鲁士蓝纳米酶标记物的制备方法及应用 |
-
2020
- 2020-06-22 CN CN202010571694.XA patent/CN111693721A/zh active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109254063A (zh) * | 2018-11-12 | 2019-01-22 | 济南大学 | 一种负载型普鲁士蓝电化学生物传感器标记物的制备方法 |
| CN110261600A (zh) * | 2019-07-02 | 2019-09-20 | 济南大学 | 一种基于四氧化三铁/普鲁士蓝纳米酶标记物的制备方法及应用 |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112945878A (zh) * | 2021-02-03 | 2021-06-11 | 安阳市妇幼保健院(安阳市儿童医院) | 间接光度法测定多巴胺的方法 |
| CN113913183A (zh) * | 2021-09-24 | 2022-01-11 | 山东师范大学 | 一种氧化型tmb纳米材料及其在检测谷胱甘肽中的应用 |
| CN113913183B (zh) * | 2021-09-24 | 2023-10-20 | 山东师范大学 | 一种氧化型tmb纳米材料及其在检测谷胱甘肽中的应用 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Chen et al. | An ultra-sensitive chemiluminescence immunosensor of carcinoembryonic antigen using HRP-functionalized mesoporous silica nanoparticles as labels | |
| US4016043A (en) | Enzymatic immunological method for the determination of antigens and antibodies | |
| CN104865247B (zh) | 基于纳米金聚集的显色方法在免疫检测中的应用 | |
| Chen et al. | A novel chemiluminescence immunoassay of staphylococcal enterotoxin B using HRP-functionalised mesoporous silica nanoparticle as label | |
| CN103913573A (zh) | 一种基于纳米金和氧化石墨烯的双信号放大elisa检测方法 | |
| Yuan et al. | A biosensor based on self-clickable AIEgen: a signal amplification strategy for ultrasensitive immunoassays | |
| WO2022152147A1 (zh) | 一种胶乳增强竞争免疫比浊检测方法及试剂盒 | |
| CN111693721A (zh) | 基于普鲁士蓝纳米酶标记物的酶联免疫吸附实验的制备方法及应用 | |
| Mao et al. | Simultaneous detection of carcinoembryonic antigen and neuron-specific enolase in human serum based on time-resolved chemiluminescence immunoassay | |
| US4971904A (en) | Heterogeneous immunoassay | |
| US9903856B2 (en) | Optical biosensor | |
| Sun et al. | A colorimetric method for determination of the prostate specific antigen based on enzyme-free cascaded signal amplification via peptide-copper (II) nanoparticles | |
| CN109239326A (zh) | 基于磁颗粒纳米酶的微流控免疫芯片分析方法及应用 | |
| CN110261600A (zh) | 一种基于四氧化三铁/普鲁士蓝纳米酶标记物的制备方法及应用 | |
| CN110967493B (zh) | 一种维生素b12测定试剂盒及其制备方法 | |
| WO2024120133A1 (zh) | 疏水性干扰物及其制备方法和应用 | |
| CN105158469B (zh) | 一种基于生物素化氨基化Fe3O4与链霉亲和素的免疫传感器的制备方法及应用 | |
| CN117554615A (zh) | 一种adamts13酶活性发光免疫检测方法及adamts13酶活性检测试剂盒 | |
| CN112763703A (zh) | 一种免疫磁珠及其制备方法和应用 | |
| CN111693689B (zh) | 一种用于酶促化学发光检测的纳米酶及其用途 | |
| CN115436335B (zh) | 一种基于苝衍生物探针免标记检测凝血酶的方法 | |
| CN113376378A (zh) | 一种d-二聚体检测试剂盒、制备方法及用途 | |
| KR20010025027A (ko) | 면역측정시약 및 면역측정법 | |
| CN112305228A (zh) | 一种肌红蛋白直接化学发光检测试剂盒、制备方法及用途 | |
| Zhan et al. | Magnetic bead-based sandwich immunoassay for viral pathogen detection by employing gold nanoparticle as carrier |
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: 20200922 |