CN111693721A - Preparation method and application of enzyme-linked immunosorbent assay based on prussian blue nano enzyme label - Google Patents
Preparation method and application of enzyme-linked immunosorbent assay based on prussian blue nano enzyme label Download PDFInfo
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- 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
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- 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
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- 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
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- 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
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- 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
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- 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)
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
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- 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
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
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Abstract
The invention discloses a preparation method and application of an enzyme-linked immunosorbent assay based on a Prussian blue nano enzyme marker, and relates to the fields of nano science, biological immunity technology, enzyme immunoassay technology and the like. The Prussian blue nano enzyme label loaded on the ferroferric oxide @ polydopamine core-shell composite material is prepared by utilizing the excellent catalytic performance of Prussian blue. By utilizing the magnetism of the ferroferric oxide ball core and the excellent biocompatibility of polydopamine, the immobilization and magnetic separation of biomolecules are realized. By utilizing the capacity of the polydopamine for reducing ferric ions under an acidic condition, the Prussian blue nano enzyme is successfully generated on the surface of the polydopamine to serve as a marker. The Prussian blue nano-enzyme catalyzes the oxidation reaction of hydrogen peroxide to change the substrate tetramethyl benzidine from colorless to blue. The marker can be suitable for preparation of various enzyme-linked immunoassays, and has wide application prospects in scientific research and clinic.
Description
Technical Field
The invention relates to the fields of nano science, biological immunity technology, enzyme immunoassay technology and the like, in particular to a preparation method and an immune chromogenic application of an enzyme-linked immune reaction marker.
Background
Biomarker detection is the only noninvasive method for early warning diseases such as cancer at present, and has very important significance for general investigation, diagnosis, prognosis judgment and the like of tumors in clinic. Among the numerous biomarker detection methods, enzyme-linked immunosorbent assay (ELISA) is a main analytical technique for quantitative detection of biomolecules due to its advantages of rapid detection, simple method, strong specificity, high sensitivity and the like.
The enzyme-linked immunosorbent assay is to specifically combine an antigen or an antibody adsorbed on a solid phase carrier with an enzyme-labeled antibody. After the substrate solution is added, the substrate changes the hydrogen donor contained therein from colorless reduced form to colored oxidized form under the action of the enzyme, and a color reaction occurs. Therefore, the presence or absence of the corresponding immunoreaction can be judged according to the color reaction of the substrate, and the shade of the color reaction is in direct proportion to the amount of the corresponding antibody or antigen in the sample. The color reaction can be quantitatively determined by an enzyme-labeling instrument, and the enzyme-linked immunosorbent assay becomes a specific and sensitive detection method by combining the specificity of the antigen-antibody reaction and the sensitivity of the enzyme chemical reaction. In view of this, the invention discloses a nano enzyme with catalytic performance as a marker for enzyme-linked immunosorbent assay, which has important practical significance.
By utilizing the magnetism of the ferroferric oxide ball core and the excellent biocompatibility of polydopamine, not only the immobilization and magnetic separation of biomolecules are realized, but also the binding capacity of the antibody on the surface of the nano-composite is greatly improved, and the preparation effect of easy labeling is realized. The prussian blue nanoenzyme is called "artificial enzyme peroxidase" because it exhibits high specificity and catalytic performance for reducing hydrogen peroxide. Different from the traditional method for preparing Prussian blue by mixing iron salt, hexacyanoferrate and iron atoms in different oxidation states, the Prussian blue nano enzyme serving as a marker can be obtained on the surface of the composite material by reducing a ferricyanide-iron ion mixture by using a ferroferric oxide @ polydopamine core-shell composite material, and the catalytic color development of the color development liquid is further realized. And the detection of the concentration of the antigen is realized by quantitative determination through a microplate reader.
The Prussian blue nano enzyme label loaded on the ferroferric oxide @ polydopamine core-shell composite material is prepared by utilizing the excellent catalytic performance of Prussian blue. By utilizing the magnetism of the ferroferric oxide ball core and the excellent biocompatibility of polydopamine, the immobilization and magnetic separation of biomolecules are realized. By utilizing the capacity of reducing ferric ions under the poly-dopamine acidic condition, the Prussian blue nano-enzyme is successfully generated on the surface of poly-dopamine to serve as a marker. The Prussian blue nano-enzyme catalyzes the oxidation reaction of hydrogen peroxide to change the substrate tetramethyl benzidine from colorless to blue.
Disclosure of Invention
One of the purposes of the invention is to provide a method for preparing prussian blue nanoenzyme on the surface of ferroferric oxide @ polydopamine core-shell composite material by utilizing polydopamine to reduce a ferricyanide-ferric ion mixture.
The second purpose of the invention is to realize the color change of the color developing solution by utilizing the catalytic action of the prussian blue on the hydrogen peroxide.
The prepared marker is used for constructing sandwich type enzyme-linked immunosorbent assay by using the labeled antibody, so that the biomarker can be efficiently and sensitively detected, and quantitative detection can be realized.
The technical scheme of the invention is as follows:
the preparation method of enzyme-linked immunosorbent assay based on Prussian blue nano enzyme label comprises the following steps:
(1) preparing ferroferric oxide nano particles: adding 0.2 g of sodium dodecyl sulfate, 1.5 g of sodium acetate and 0.5 g of ferric chloride into 15 mL of ethylene glycol, and stirring for 30 minutes at room temperature; then transferring the mixed solution into a polytetrafluoroethylene high-temperature reaction kettle and heating for 12 hours at 200 ℃; washing the obtained product with ultrapure water and ethanol for three times respectively; the final product was dried under vacuum at 35 ℃ for 12 hours; drying, grinding and storing at room temperature;
(2) preparing a ferroferric oxide @ polydopamine core-shell composite material: adding 100 mg of ferroferric oxide nanoparticles and 200mg of dopamine into a mixed solution of 120 mL of Tris-HCl buffer solution with the pH =8.8 and 100 mL of isopropanol; stirring for 13 hours to obtain a black suspension; the product was collected by magnetic separation and washed five times with ultrapure water to remove unreacted materials, and then dried in vacuum at 35 ℃; weighing after drying, re-dispersing in 5 mL of ultrapure water, and refrigerating and storing in a refrigerator at 4 ℃;
(3) taking 5 mL of 0.1 mol.L of 50 mu L ferroferric oxide @ polydopamine core-shell composite dispersion liquid-1And pH 7.4, and 2 mL of a 10. mu.g/mL phosphate buffer solution was added thereto-1Incubating the procalcitonin detection antibody solution at 4 ℃ for 2h, and centrifuging; adding 100 μ L of 0.1% bovine serum albumin solution to block nonspecific binding site, incubating at 4 deg.C for 2 hr, centrifuging to obtain solid product, and dispersing in 5 mL of 0.1 mol/L-1Preparing a procalcitonin antibody solution marked by ferroferric oxide @ polydopamine core-shell composite material in a phosphate buffer solution with the pH of 7.4;
(4) mixing procalcitonin antibody solution marked by ferroferric oxide @ polydopamine core-shell composite material with 0.1 ng/mL-1Mixing the antigens in a volume ratio of 1:1, incubating for 1h at room temperature, and performing centrifugal separation to obtain a procalcitonin antibody solution labeled by ferroferric oxide @ polydopamine core-shell composite material specifically bound with the antigens;
(5) add 100. mu.L, 1. mu.g.mL to 96 microwell plate-1Antibody solution of procalcitonin, incubating at 4 deg.C for 12 hr, and buffering with phosphateWashing the microporous plate for three times by using the flushing liquid; then adding 100 mu L of 0.1 percent bovine serum albumin solution to seal the nonspecific binding sites on the microplate, incubating for 1h at room temperature, and washing the plate for three times by using phosphate buffer solution; adding a procalcitonin antibody solution labeled by ferroferric oxide @ polydopamine core-shell composite material specifically bound with the antigen, incubating for 1h, and washing the plate with ultrapure water for three times; adding 200 μ L, 1 mmol. multidot.L into the micropore-1Reacting the mixed solution of ferric trichloride and potassium ferricyanide for 1min, removing the mixed iron solution, washing the plate with ultrapure water for three times, and preparing the marker loaded with the Prussian blue nano-enzyme;
(6) the immune color development process comprises the following steps: adding 100 mu L of microplate successfully loaded with Prussian blue nano enzyme label with the concentration of 1.248 mmol.L-1100 mu L of 3,3',5,5' -tetramethylbenzidine color developing solution with the concentration of 6 mmol.L-1The solution in the microplate is changed from colorless to blue; the developing time is 15 min, and 50 μ L of 1 mol/L is added after the development is finished-1The solution in the microporous plate is changed from blue to yellow; and measuring the absorbance of the solution of 350 nm-900nm by using a microplate reader.
Advantageous results of the invention
Compared with the prior art, the invention has the following advantages:
1) the Prussian blue nano-enzyme with excellent catalytic performance is prepared by reducing ferricyanide-ferric ion mixture with polydopamine, and the preparation method is simple;
2) the marker prepared by the method realizes the color change of the color development liquid by utilizing the catalytic action of the prussian blue nano enzyme on hydrogen peroxide, so that the efficiency of an enzyme-linked immunosorbent assay is greatly improved;
3) the marker prepared by the method utilizes ferroferric oxide @ polydopamine core-shell composite material to load immune substances, so that the preparation effect of easy marking is achieved.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1
Preparation method of Prussian blue nano enzyme label
(1) Preparing ferroferric oxide nano particles: adding 0.2 g of sodium dodecyl sulfate, 1.5 g of sodium acetate and 0.5 g of ferric chloride into 15 mL of ethylene glycol, and stirring for 30 minutes at room temperature; then transferring the mixed solution into a polytetrafluoroethylene high-temperature reaction kettle and heating for 12 hours at 200 ℃; washing the obtained product with ultrapure water and ethanol for three times respectively; the final product was dried under vacuum at 35 ℃ for 12 hours; drying, grinding and storing at room temperature;
(2) preparing a ferroferric oxide @ polydopamine core-shell composite material: adding 100 mg of ferroferric oxide nanoparticles and 200mg of dopamine into a mixed solution of 120 mL of Tris-HCl buffer solution with the pH =8.8 and 100 mL of isopropanol; stirring for 13 hours to obtain a black suspension; the product was collected by magnetic separation and washed five times with ultrapure water to remove unreacted materials, and then dried in vacuum at 35 ℃; weighing after drying, re-dispersing in 5 mL of ultrapure water, and refrigerating and storing in a refrigerator at 4 ℃;
(3) taking 5 mL of 0.1 mol.L of 50 mu L ferroferric oxide @ polydopamine core-shell composite dispersion liquid-1And pH 7.4, and 2 mL of a 10. mu.g/mL phosphate buffer solution was added thereto-1Incubating the procalcitonin detection antibody solution at 4 ℃ for 2h, and centrifuging; adding 100 μ L of 0.1% bovine serum albumin solution to block nonspecific binding site, incubating at 4 deg.C for 2 hr, centrifuging to obtain solid product, and dispersing in 5 mL of 0.1 mol/L-1Preparing a procalcitonin antibody solution marked by ferroferric oxide @ polydopamine core-shell composite material in a phosphate buffer solution with the pH of 7.4;
(4) mixing procalcitonin antibody solution marked by ferroferric oxide @ polydopamine core-shell composite material with 0.1 ng/mL-1Mixing the antigens in a volume ratio of 1:1, incubating for 1h at room temperature, and performing centrifugal separation to obtain a procalcitonin antibody solution labeled by ferroferric oxide @ polydopamine core-shell composite material specifically bound with the antigens;
(5) add 100. mu.L, 1. mu.g.mL to 96 microwell plate-1Incubating the antibody solution of procalcitonin at 4 ℃ for 12h, and washing the microporous plate with a phosphate buffer solution for three times; then adding 100 mu L of 0.1 percent bovine serum albumin solution to seal the nonspecific binding sites on the microplate, incubating for 1h at room temperature, and washing the plate for three times by using phosphate buffer solution; adding a procalcitonin antibody solution labeled by ferroferric oxide @ polydopamine core-shell composite material specifically bound with the antigen, incubating for 1h, and washing the plate with ultrapure water for three times; adding 200 μ L, 1 mmol. multidot.L into the micropore-1Reacting the mixed solution of ferric trichloride and potassium ferricyanide for 1min, removing the mixed iron solution, washing the plate with ultrapure water for three times, and preparing the marker loaded with the Prussian blue nano-enzyme.
Example 2
Construction of sandwich type procalcitonin enzyme-linked immunosorbent assay
(1) Coating: add 100. mu.L, 1. mu.g.mL to 96 microwell plate-1Incubating procalcitonin antibody solution at 4 ℃ for 12 h; washing the plate with phosphate buffer solution for three times, and spin-drying;
(2) and (3) sealing: blocking the non-specific binding sites with 100 μ L of 0.1% bovine serum albumin solution, incubating at 37 deg.C for 1 h;
(3) sample adding: throwing off confining liquid, adding a procalcitonin antibody solution labeled by ferroferric oxide @ polydopamine core-shell composite material specifically bound with antigen, and incubating for 1h at 37 ℃; washing the plate with phosphate buffer solution for three times, and spin-drying;
(3) nano-enzyme: adding 200 mu L of mixed solution of ferric trichloride and potassium ferricyanide into the micropores, reacting for 1min, removing iron liquid, and washing the plate with ultrapure water for three times;
(4) color development: adding 100 mu L of color developing agent A, B into each hole (color developing solution A: 2.72 g of sodium acetate, 0.3 g of citric acid, 60 mu L of 30% hydrogen peroxide, distilled water to 100 mL, color developing solution B: 0.04 g of disodium ethylenediamine tetraacetic acid, 0.19 g of citric acid and 10 mL of glycerol), dissolving 0.03 g of tetramethylbenzidine in 0.6 mL of dimethyl sulfoxide, adding a small amount of water, stirring in dark place to dissolve, and fixing the volume to 100 mL);
(5) and (4) terminating:50 μ L of 1 mol. L was added to each well-1The sulfuric acid stop solution;
(6) and (3) detection: the absorbance at 450 nm was measured on a TECAN microplate reader.
Claims (4)
1. The preparation method of enzyme-linked immunosorbent assay based on Prussian blue nano enzyme label is characterized by comprising the following steps:
(1) preparing ferroferric oxide nano particles: adding 0.2 g of sodium dodecyl sulfate, 1.5 g of sodium acetate and 0.5 g of ferric chloride into 15 mL of ethylene glycol, and stirring for 30 minutes at room temperature; then transferring the mixed solution into a polytetrafluoroethylene high-temperature reaction kettle and heating for 12 hours at 200 ℃; washing the obtained product with ultrapure water and ethanol for three times respectively; the final product was dried under vacuum at 35 ℃ for 12 hours; drying, grinding and storing at room temperature;
(2) preparing a ferroferric oxide @ polydopamine core-shell composite material: adding 100 mg of ferroferric oxide nanoparticles and 200mg of dopamine into a mixed solution of 120 mL of Tris-HCl buffer solution with the pH =8.8 and 100 mL of isopropanol; stirring for 13 hours to obtain a black suspension; the product was collected by magnetic separation and washed five times with ultrapure water to remove unreacted materials, and then dried in vacuum at 35 ℃; weighing after drying, re-dispersing in 5 mL of ultrapure water, and refrigerating and storing in a refrigerator at 4 ℃;
(3) taking 5 mL of 0.1 mol.L of 50 mu L ferroferric oxide @ polydopamine core-shell composite dispersion liquid-1And pH 7.4, and 2 mL of a 10. mu.g/mL phosphate buffer solution was added thereto-1Incubating the procalcitonin detection antibody solution at 4 ℃ for 2h, and centrifuging; adding 100 μ L of 0.1% bovine serum albumin solution to block nonspecific binding site, incubating at 4 deg.C for 2 hr, centrifuging to obtain solid product, and dispersing in 5 mL of 0.1 mol/L-1Preparing a procalcitonin antibody solution marked by ferroferric oxide @ polydopamine core-shell composite material in a phosphate buffer solution with the pH of 7.4;
(4) dissolving procalcitonin antibody marked by ferroferric oxide @ polydopamine core-shell composite materialMixing the solution with 0.1 ng/mL-1Mixing the antigens in a volume ratio of 1:1, incubating for 1h at room temperature, and performing centrifugal separation to obtain a procalcitonin antibody solution labeled by ferroferric oxide @ polydopamine core-shell composite material specifically bound with the antigens;
(5) add 100. mu.L, 1. mu.g.mL to 96 microwell plate-1Incubating the antibody solution of procalcitonin at 4 ℃ for 12h, and washing the microporous plate with a phosphate buffer solution for three times; then adding 100 mu L of 0.1 percent bovine serum albumin solution to seal the nonspecific binding sites on the microplate, incubating for 1h at room temperature, and washing the plate for three times by using phosphate buffer solution; adding a procalcitonin antibody solution labeled by ferroferric oxide @ polydopamine core-shell composite material specifically bound with the antigen, incubating for 1h, and washing the plate with ultrapure water for three times; adding 200 μ L, 1 mmol. multidot.L into the micropore-1Reacting the mixed solution of ferric trichloride and potassium ferricyanide for 1min, removing the mixed iron solution, washing the plate with ultrapure water for three times, and preparing the marker loaded with the Prussian blue nano-enzyme;
(6) the immune color development process comprises the following steps: adding 100 mu L of microplate successfully loaded with Prussian blue nano enzyme label with the concentration of 1.248 mmol.L-1100 mu L of 3,3',5,5' -tetramethylbenzidine color developing solution with the concentration of 6 mmol.L-1The solution in the microplate is changed from colorless to blue; the developing time is 15 min, and 50 μ L of 1 mol/L is added after the development is finished-1The solution in the microporous plate is changed from blue to yellow; and measuring the absorbance of the solution of 350 nm-900nm by using a microplate reader.
2. The method for preparing the prussian blue nano-enzyme label according to claim 1, wherein the label is used for catalytic development of a developing solution.
3. The use of the prepared prussian blue nano-enzyme label according to claim 1 as an enzyme-linked immunosorbent assay for procalcitonin detection.
4. The use of an enzyme-linked immunosorbent assay according to claim 3 as a procalcitonin assay, characterized in that the detection steps are as follows:
(1) coating: add 100. mu.L, 1. mu.g.mL to 96 microwell plate-1Incubating procalcitonin antibody solution at 4 ℃ for 12 h; washing the plate with phosphate buffer solution for three times, and spin-drying;
(2) and (3) sealing: sealing with 100 μ L of sealing solution, and incubating at 37 deg.C for 1 h;
(3) sample adding: throwing off confining liquid, adding a procalcitonin antibody solution labeled by ferroferric oxide @ polydopamine core-shell composite material specifically bound with antigen, and incubating for 1h at 37 ℃; washing the plate with phosphate buffer solution for three times, and spin-drying;
(3) nano-enzyme: adding 200 uL of mixed solution of ferric trichloride and potassium ferricyanide into the micropores, reacting for 1min, removing iron liquid, and washing the plate with ultrapure water for three times;
(4) color development: adding 100 mu L of color developing agent A, B into each well in sequence;
(5) and (4) terminating: adding 50 mu L of stop solution into each hole;
(6) and (3) detection: the absorbance at 450 nm was measured on a TECAN microplate reader.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112945878A (en) * | 2021-02-03 | 2021-06-11 | 安阳市妇幼保健院(安阳市儿童医院) | Method for measuring dopamine by indirect photometry |
| CN113913183A (en) * | 2021-09-24 | 2022-01-11 | 山东师范大学 | Oxidized TMB nano material and application thereof in detection of glutathione |
| CN114791488A (en) * | 2022-02-18 | 2022-07-26 | 华南农业大学 | Probe platform and method for detecting salbutamol based on magnetic-assisted Prussian blue nanoenzyme-DAB substrate |
| CN115856193A (en) * | 2022-12-07 | 2023-03-28 | 中国科学院合肥物质科学研究院 | Copper-chromium-doped Prussian blue nano-enzyme, preparation method thereof and application thereof in rapid detection of glyphosate |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109254063A (en) * | 2018-11-12 | 2019-01-22 | 济南大学 | A kind of preparation method of the Prussian blue electrochemica biological sensor marker of support type |
| CN110261600A (en) * | 2019-07-02 | 2019-09-20 | 济南大学 | It is a kind of based on ferroso-ferric oxide/prussian blue nano enzyme marker preparation method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109254063A (en) * | 2018-11-12 | 2019-01-22 | 济南大学 | A kind of preparation method of the Prussian blue electrochemica biological sensor marker of support type |
| CN110261600A (en) * | 2019-07-02 | 2019-09-20 | 济南大学 | It is a kind of based on ferroso-ferric oxide/prussian blue nano enzyme marker preparation method and application |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112945878A (en) * | 2021-02-03 | 2021-06-11 | 安阳市妇幼保健院(安阳市儿童医院) | Method for measuring dopamine by indirect photometry |
| CN113913183A (en) * | 2021-09-24 | 2022-01-11 | 山东师范大学 | Oxidized TMB nano material and application thereof in detection of glutathione |
| CN113913183B (en) * | 2021-09-24 | 2023-10-20 | 山东师范大学 | Oxidized TMB nano material and application thereof in detection of glutathione |
| CN114791488A (en) * | 2022-02-18 | 2022-07-26 | 华南农业大学 | Probe platform and method for detecting salbutamol based on magnetic-assisted Prussian blue nanoenzyme-DAB substrate |
| CN115856193A (en) * | 2022-12-07 | 2023-03-28 | 中国科学院合肥物质科学研究院 | Copper-chromium-doped Prussian blue nano-enzyme, preparation method thereof and application thereof in rapid detection of glyphosate |
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