CN116068163B - Method and kit for detecting interferon alpha 2b - Google Patents
Method and kit for detecting interferon alpha 2b Download PDFInfo
- Publication number
- CN116068163B CN116068163B CN202310133265.8A CN202310133265A CN116068163B CN 116068163 B CN116068163 B CN 116068163B CN 202310133265 A CN202310133265 A CN 202310133265A CN 116068163 B CN116068163 B CN 116068163B
- Authority
- CN
- China
- Prior art keywords
- interferon alpha
- solution
- antibody
- incubation
- alpha
- 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.)
- Active
Links
- 102100040018 Interferon alpha-2 Human genes 0.000 title claims abstract description 84
- 108010079944 Interferon-alpha2b Proteins 0.000 title claims abstract description 84
- 238000000034 method Methods 0.000 title claims abstract description 28
- 229920002873 Polyethylenimine Polymers 0.000 claims abstract description 121
- 102000003992 Peroxidases Human genes 0.000 claims abstract description 50
- 238000001514 detection method Methods 0.000 claims abstract description 50
- 108040007629 peroxidase activity proteins Proteins 0.000 claims abstract description 49
- 238000011534 incubation Methods 0.000 claims abstract description 43
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical class O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 claims abstract description 34
- 239000002105 nanoparticle Substances 0.000 claims abstract description 34
- 150000002978 peroxides Chemical class 0.000 claims abstract description 29
- 238000002835 absorbance Methods 0.000 claims abstract description 22
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 16
- 239000000243 solution Substances 0.000 claims description 56
- 238000011161 development Methods 0.000 claims description 25
- 239000007788 liquid Substances 0.000 claims description 24
- 102000004190 Enzymes Human genes 0.000 claims description 18
- 108090000790 Enzymes Proteins 0.000 claims description 18
- ZNZYKNKBJPZETN-WELNAUFTSA-N Dialdehyde 11678 Chemical compound N1C2=CC=CC=C2C2=C1[C@H](C[C@H](/C(=C/O)C(=O)OC)[C@@H](C=C)C=O)NCC2 ZNZYKNKBJPZETN-WELNAUFTSA-N 0.000 claims description 17
- 239000007853 buffer solution Substances 0.000 claims description 16
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical group OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 12
- 238000002156 mixing Methods 0.000 claims description 12
- 150000003839 salts Chemical class 0.000 claims description 12
- 238000004729 solvothermal method Methods 0.000 claims description 10
- 239000004094 surface-active agent Substances 0.000 claims description 10
- 230000000903 blocking effect Effects 0.000 claims description 9
- 239000003638 chemical reducing agent Substances 0.000 claims description 9
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 8
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical group OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 7
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 claims description 6
- 230000035484 reaction time Effects 0.000 claims description 5
- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims description 4
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 4
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 4
- 239000004372 Polyvinyl alcohol Substances 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
- KTWOOEGAPBSYNW-UHFFFAOYSA-N ferrocene Chemical compound [Fe+2].C=1C=C[CH-]C=1.C=1C=C[CH-]C=1 KTWOOEGAPBSYNW-UHFFFAOYSA-N 0.000 claims description 3
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 3
- 239000011790 ferrous sulphate Substances 0.000 claims description 3
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 3
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 3
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 3
- 235000019422 polyvinyl alcohol Nutrition 0.000 claims description 3
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 3
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 3
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 3
- 235000017281 sodium acetate Nutrition 0.000 claims description 3
- 239000001632 sodium acetate Substances 0.000 claims description 3
- 235000019333 sodium laurylsulphate Nutrition 0.000 claims description 3
- 230000035945 sensitivity Effects 0.000 abstract description 10
- 238000003018 immunoassay Methods 0.000 abstract description 9
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 109
- 230000000694 effects Effects 0.000 description 20
- 239000000872 buffer Substances 0.000 description 18
- 108010047761 Interferon-alpha Proteins 0.000 description 16
- 102000006992 Interferon-alpha Human genes 0.000 description 16
- 238000006243 chemical reaction Methods 0.000 description 16
- OHDXDNUPVVYWOV-UHFFFAOYSA-N n-methyl-1-(2-naphthalen-1-ylsulfanylphenyl)methanamine Chemical compound CNCC1=CC=CC=C1SC1=CC=CC2=CC=CC=C12 OHDXDNUPVVYWOV-UHFFFAOYSA-N 0.000 description 16
- 230000003197 catalytic effect Effects 0.000 description 14
- 210000002966 serum Anatomy 0.000 description 14
- 238000002965 ELISA Methods 0.000 description 13
- 238000012986 modification Methods 0.000 description 10
- 230000004048 modification Effects 0.000 description 10
- 238000005406 washing Methods 0.000 description 10
- 238000010521 absorption reaction Methods 0.000 description 8
- 230000008859 change Effects 0.000 description 8
- 101000693922 Bos taurus Albumin Proteins 0.000 description 6
- 230000005291 magnetic effect Effects 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 206010028980 Neoplasm Diseases 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 238000011156 evaluation Methods 0.000 description 5
- 239000002086 nanomaterial Substances 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 241000282326 Felis catus Species 0.000 description 4
- 108010002350 Interleukin-2 Proteins 0.000 description 4
- 102000000588 Interleukin-2 Human genes 0.000 description 4
- 238000002441 X-ray diffraction Methods 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 150000002505 iron Chemical class 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical group CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 108090000978 Interleukin-4 Proteins 0.000 description 3
- 108090001005 Interleukin-6 Proteins 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 238000006555 catalytic reaction Methods 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 230000008878 coupling Effects 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000002296 dynamic light scattering Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 230000005415 magnetization Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- NMWKYTGJWUAZPZ-WWHBDHEGSA-N (4S)-4-[[(4R,7S,10S,16S,19S,25S,28S,31R)-31-[[(2S)-2-[[(1R,6R,9S,12S,18S,21S,24S,27S,30S,33S,36S,39S,42R,47R,53S,56S,59S,62S,65S,68S,71S,76S,79S,85S)-47-[[(2S)-2-[[(2S)-4-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-3-methylbutanoyl]amino]-3-methylbutanoyl]amino]-3-hydroxypropanoyl]amino]-3-(1H-imidazol-4-yl)propanoyl]amino]-3-phenylpropanoyl]amino]-4-oxobutanoyl]amino]-3-carboxypropanoyl]amino]-18-(4-aminobutyl)-27,68-bis(3-amino-3-oxopropyl)-36,71,76-tribenzyl-39-(3-carbamimidamidopropyl)-24-(2-carboxyethyl)-21,56-bis(carboxymethyl)-65,85-bis[(1R)-1-hydroxyethyl]-59-(hydroxymethyl)-62,79-bis(1H-imidazol-4-ylmethyl)-9-methyl-33-(2-methylpropyl)-8,11,17,20,23,26,29,32,35,38,41,48,54,57,60,63,66,69,72,74,77,80,83,86-tetracosaoxo-30-propan-2-yl-3,4,44,45-tetrathia-7,10,16,19,22,25,28,31,34,37,40,49,55,58,61,64,67,70,73,75,78,81,84,87-tetracosazatetracyclo[40.31.14.012,16.049,53]heptaoctacontane-6-carbonyl]amino]-3-methylbutanoyl]amino]-7-(3-carbamimidamidopropyl)-25-(hydroxymethyl)-19-[(4-hydroxyphenyl)methyl]-28-(1H-imidazol-4-ylmethyl)-10-methyl-6,9,12,15,18,21,24,27,30-nonaoxo-16-propan-2-yl-1,2-dithia-5,8,11,14,17,20,23,26,29-nonazacyclodotriacontane-4-carbonyl]amino]-5-[[(2S)-1-[[(2S)-1-[[(2S)-3-carboxy-1-[[(2S)-1-[[(2S)-1-[[(1S)-1-carboxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-3-(1H-imidazol-4-yl)-1-oxopropan-2-yl]amino]-5-oxopentanoic acid Chemical compound CC(C)C[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](Cc1c[nH]cn1)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H]1CSSC[C@H](NC(=O)[C@@H](NC(=O)[C@@H]2CSSC[C@@H]3NC(=O)[C@H](Cc4ccccc4)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](Cc4c[nH]cn4)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H]4CCCN4C(=O)[C@H](CSSC[C@H](NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](Cc4c[nH]cn4)NC(=O)[C@H](Cc4ccccc4)NC3=O)[C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](Cc3ccccc3)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N3CCC[C@H]3C(=O)N[C@@H](C)C(=O)N2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](Cc2ccccc2)NC(=O)[C@H](Cc2c[nH]cn2)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)C(C)C)[C@@H](C)O)C(C)C)C(=O)N[C@@H](Cc2c[nH]cn2)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](Cc2ccc(O)cc2)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1)C(=O)N[C@@H](C)C(O)=O NMWKYTGJWUAZPZ-WWHBDHEGSA-N 0.000 description 2
- 102000014150 Interferons Human genes 0.000 description 2
- 108010050904 Interferons Proteins 0.000 description 2
- 102000006747 Transforming Growth Factor alpha Human genes 0.000 description 2
- 101800004564 Transforming growth factor alpha Proteins 0.000 description 2
- 208000036142 Viral infection Diseases 0.000 description 2
- 238000000862 absorption spectrum Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 229940079322 interferon Drugs 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 238000012933 kinetic analysis Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000001878 scanning electron micrograph Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 238000004627 transmission electron microscopy Methods 0.000 description 2
- 238000001291 vacuum drying Methods 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 229910017135 Fe—O Inorganic materials 0.000 description 1
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 1
- 238000001157 Fourier transform infrared spectrum Methods 0.000 description 1
- 206010020843 Hyperthermia Diseases 0.000 description 1
- 108010078049 Interferon alpha-2 Proteins 0.000 description 1
- 206010029350 Neurotoxicity Diseases 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 108700020962 Peroxidase Proteins 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 238000003917 TEM image Methods 0.000 description 1
- 206010044221 Toxic encephalopathy Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- -1 amino, hydroxyl Chemical group 0.000 description 1
- 229940040526 anhydrous sodium acetate Drugs 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000001028 anti-proliverative effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 230000002155 anti-virotic effect Effects 0.000 description 1
- 238000005452 bending Methods 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 229920006317 cationic polymer Polymers 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000003593 chromogenic compound Substances 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 239000007822 coupling agent Substances 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000000840 electrochemical analysis Methods 0.000 description 1
- 238000000835 electrochemical detection Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 230000005294 ferromagnetic effect Effects 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000036031 hyperthermia Effects 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000002595 magnetic resonance imaging Methods 0.000 description 1
- 238000007885 magnetic separation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 230000007135 neurotoxicity Effects 0.000 description 1
- 231100000228 neurotoxicity Toxicity 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000004983 pleiotropic effect Effects 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 239000011241 protective layer Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000004626 scanning electron microscopy Methods 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000004416 surface enhanced Raman spectroscopy Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 230000029812 viral genome replication Effects 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Hematology (AREA)
- General Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a detection method of interferon alpha 2b, which comprises the following steps: dripping a sample to be detected of interferon alpha 2b into a detection plate coated with an interferon alpha 2b antibody for first incubation, adding peroxidase coupled with the interferon alpha 2b antibody for second incubation, adding an auxiliary reagent for third incubation, and detecting absorbance; the peroxide nano-enzyme is a polyethyleneimine modified ferroferric oxide nano-particle. Compared with the prior art, the method has the advantages of strong antibody capturing capability, high specificity and sensitivity and low cost by taking the polyethyleneimine modified ferroferric oxide nano-particles as the peroxide nano-enzyme to carry out immunoassay on the interferon alpha 2 b.
Description
Technical Field
The invention belongs to the technical field of enzyme-linked immunosorbent assay, and particularly relates to a detection method and a detection kit for interferon alpha 2 b.
Background
The interferon is a pleiotropic cytokine and has the functions of immunoregulation, antivirus, anticancer cell proliferation and the like. Among them, interferon alpha 2b (IFN-. Alpha.2b) has a wide range of biological activities such as inhibition of viral replication, antiproliferation, immunomodulation, and antitumor (J Pharm Biomed Anal2014,88,123-9). Because of the above characteristics, IFN-. Alpha.2b is used as one of drugs for treating virus-related diseases and for treating cancer. Optimizing pharmacokinetic monitoring of IFN-alpha 2b is advantageous for maximizing its therapeutic effect and reducing side effects such as fever, pain, and even neurotoxicity associated with interferon therapy (Nat Rev Drug Discov,2007,6,975). Therefore, quantitative studies on IFN-. Alpha.2b are of great research interest in the treatment of tumor and viral infection diseases.
However, some of the commonly used analytical methods for IFN-. Alpha.2b detection, such as electrochemical analysis, bioassays, gel electrophoresis, chromatography, etc., require expensive instruments and specialized laboratory personnel to operate, resulting in limited popularity. Although the traditional enzyme-linked immunosorbent assay (ELISA) has good sensitivity (Eur J Gynaecol Oncol 2021,42,96;Appl Biochem Biotechnol 2019,188,72) for IFN-alpha 2b detection, the traditional ELISA has the defects of high price, low stability, short shelf life, complex separation and purification and the like because of the participation of natural enzymes. Therefore, it is necessary to design other stable, easy-to-synthesize and low-cost alternative enzymes to replace the natural enzymes for the detection of IFN-. Alpha.2b, and thus for ELISA.
The nano-enzyme can simulate the catalytic property of the natural enzyme, and has been paid attention to by more and more scientific researchers because of the advantages of simple preparation method, relatively good stability, low cost, easy separation and the like. In 2007, yan et al for the first time found that ferromagnetic nanoparticles (Fe 3O4 MNPs) were able to mimic the natural peroxidase catalytic activity (Nat Nanotechnol,2007,2,577). In recent years, fe 3O4 MNPs nanoparticles are widely used in many fields, particularly in biomedical fields, including cancer hyperthermia, magnetic resonance imaging, gene transfer, targeted drug transfer, enzyme immobilization, immunoassay, etc., as alternatives to natural peroxidases, fe 3O4 MNPs have been used in biological detection methods such as ELISA (J Colloid INTERFACE SCI,2013,405,291-295;Anal Chim Acta,2021,1184,339037), electrochemical detection (Biosens Bioelectron,2022,197,113758), fluorescence detection (Sens Actuators B Chem,2021,346,130434), and Surface Enhanced Raman Spectroscopy (SERS) (Talanta, 2021,232,122432). However, in the design of ELISA immunoassays biosensors, the nanomaterials must undergo a series of modifications before they can be successfully coupled to the antibodies. In general, for convenient and efficient binding of antibodies, the nanomaterial preferably contains aldehyde, amino, hydroxyl, carboxylic acid, or other functional groups. Silicon and its derivatives and polymers are commonly used to modify nanomaterials to aid in the modification of antibodies. However, many current methods of additional coating or modification of nanomaterials suffer from the disadvantages of cumbersome process and/or poor repeatability.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a method and a kit for detecting interferon alpha 2b, wherein the method has the advantages of strong antibody capturing capability, high specificity and sensitivity, and low cost.
The invention provides a detection method of interferon alpha 2b, which comprises the following steps:
dripping a sample to be detected of interferon alpha 2b into a detection plate coated with an interferon alpha 2b antibody for first incubation, adding peroxidase coupled with the interferon alpha 2b antibody for second incubation, adding an auxiliary reagent for third incubation, and detecting absorbance;
the peroxide nano-enzyme is a polyethyleneimine modified ferroferric oxide nano-particle.
Preferably, the polyethyleneimine modified ferroferric oxide nanoparticle is prepared according to the following method:
And carrying out solvothermal reaction on ferric salt and polyethyleneimine in the presence of a reducing agent and a surfactant to obtain polyethyleneimine modified ferroferric oxide nano particles.
Preferably, the reducing agent is ethylene glycol; the surfactant is one or more of hydrazine, polyvinyl alcohol, sodium acetate, polyvinylpyrrolidone and sodium dodecyl sulfate; the ferric salt is selected from one or more of ferric chloride, ferrous sulfate and ferrocene; the mass ratio of the ferric salt to the polyethyleneimine is 1: (0.5-2); the mass ratio of the ferric salt to the surfactant is 1: (2-6); the temperature of the solvothermal reaction is 150-240 ℃; the solvothermal reaction time is 1-3 h.
Preferably, the peroxidase conjugated interferon alpha 2b antibody is prepared according to the following steps:
Mixing and reacting the peroxide nano-enzyme with dialdehyde solution to obtain dialdehyde-functionalized peroxide nano-enzyme;
And mixing the dialdehyde functionalized peroxidase with the interferon alpha 2b antibody solution, and then adding a blocking solution to block to obtain the peroxidase coupled with the interferon alpha 2b antibody.
Preferably, the dialdehyde solution is glutaraldehyde solution; the concentration of the dialdehyde solution is 5% -10% (w/v); the proportion of the peroxide nano enzyme to the dialdehyde solution is 1mg: (80-150) mu L; the time of the mixing reaction is 3-6 h.
Preferably, the mass ratio of the interferon alpha 2b antibody to the peroxidase nano-enzyme in the interferon alpha 2b antibody solution is (0.1-0.4): 1.
Preferably, the temperature of the first incubation is 36-37 ℃; the first incubation time is 0.5-1.5 h;
The temperature of the second incubation is 36-37 ℃; the second incubation time is 10-20 min;
The temperature of the third incubation is 30-45 ℃; the third incubation time is 10-20 min;
the wavelength of the detected absorbance was 652nm.
The invention also provides a detection kit of interferon alpha 2b, which comprises: an interferon alpha 2b antibody coated detection plate, a peroxide nano enzyme coupled with the interferon alpha 2b antibody and an auxiliary reagent;
the peroxide nano-enzyme is a polyethyleneimine modified ferroferric oxide nano-particle.
Preferably, the auxiliary reagent comprises a color developing solution and a buffer solution; the color development liquid comprises color development liquid A and color development liquid B.
The invention also provides application of the peroxide nano-enzyme in interferon alpha 2b detection, wherein the peroxide nano-enzyme is a polyethyleneimine modified ferroferric oxide nano-particle.
The invention provides a detection method of interferon alpha 2b, which comprises the following steps: dripping a sample to be detected of interferon alpha 2b into a detection plate coated with an interferon alpha 2b antibody for first incubation, adding peroxidase coupled with the interferon alpha 2b antibody for second incubation, adding an auxiliary reagent for third incubation, and detecting absorbance; the peroxide nano-enzyme is a polyethyleneimine modified ferroferric oxide nano-particle. Compared with the prior art, the method has the advantages of strong antibody capturing capability, high specificity and sensitivity and low cost by taking the polyethyleneimine modified ferroferric oxide nano-particles as the peroxide nano-enzyme to carry out immunoassay on the interferon alpha 2 b.
Experimental results show that the IFN-alpha 2b quantitative colorimetric method based on Fe 3O4 @PEIPIPs provided by the invention has the advantages of strong antibody capturing capability, high specificity and sensitivity and low cost, and the detection linear range is 0.075-25 ng/mL, and the detection limit is as low as 0.037ng/mL. This new immunoassay strategy has great potential for clinical application in monitoring the pharmacokinetics of IFN- α2b during viral infection and cancer treatment.
Drawings
FIG. 1 is a representation of the Fe 3O4 @PEI MNPs material prepared in example 1 of the present invention, wherein a, b is TEM of Fe 3O4 @PEI MNPs at different magnifications, c, d is an SEM image, e is an X-ray diffraction pattern (XRD) of Fe 3O4 and Fe 3O4 @PEI MNPs, and f is an infrared spectrum (FTIR); g is an optical image, and h and i are magnetic measurement images;
FIG. 2 is a graph showing the sensitivity contrast of Fe 3O4 @PEIPPs prepared from PEI with different weight average molecular weights in example 1 of the present invention for detecting IFN-. Alpha.2b with different concentrations;
FIG. 3 is an identification of IFN-. Alpha.2b antibodies coupled to Fe 3O4 @PEIPMNPs in example 1 of the invention, wherein a is the Zeta potential diagram of Fe 3O4MNPs、Fe3O4 @PEIPMNPs and Fe 3O4 @PEI-anti body, b is the hydration mechanical dimension diagram of Fe 3O4 @PEIPMNPs before and after coupling with antibodies, and c is the ultraviolet-visible absorption diagram of Fe 3O4@PEI MNPs、Fe3O4 @PEI-anti body and IFN-. Alpha.2b antibodies;
FIG. 4 is a graph showing the measurement result of the peroxidase activity of Fe 3O4 @PEI MNPs in example 1 of the present invention, wherein a is an ultraviolet-visible absorption spectrum of TMB under different conditions, and b is a graph showing the time-dependent absorbance change of Fe 3O4 @PEI MNPs or Fe 3O4 @PEI-anti body catalyzed at 652 nm;
FIG. 5 is a graph showing the evaluation of peroxidase activity under different conditions of Fe 3O4 @PEI MNPs in example 1 of the present invention, wherein a is a graph showing the evaluation of peroxidase activity at different concentrations of Fe 3O4 @PEI MNPs, b is a graph showing the evaluation of peroxidase activity at different pH values, c is a graph showing the evaluation of peroxidase activity at different temperatures, and d is a graph showing the evaluation of peroxidase activity at different concentrations of H 2O2;
FIG. 6 is a graph of steady state kinetics analysis and Lineweaver-Burk of peroxidase activity for Fe 3O4 @PEI MNPs in example 1 of the present invention, where a is a Michaelis-Menten plot of 1.25M H 2O2 versus TMB at different concentrations versus H 2O2 at different concentrations, b is a Michaelis-Menten plot of 832 μM TMB versus H 2O2 at different concentrations, c is a double reciprocal plot of TMB versus v corresponding to the Michaelis-Menten plot, and d is a double reciprocal plot of H 2O2 versus v;
FIG. 7 is a graph showing the results of the study of Fe 3O4 @PEIPIPs for IFN-. Alpha.2b detection in example 1 of the present invention, wherein a is a graph of absorbance (ΔA) versus IFN-. Alpha.2b concentration at 652nm, and b is a standard graph of Log ΔA versus Log C for different IFN-. Alpha.2b concentrations;
FIG. 8 is a graph showing the results of the detection of IFN-. Alpha.2b in human serum under the condition of a 1. Mu.g/mL concentration of IFN-. Alpha.2b (10 ng/mL), IL-2, IL-4, IL-6 and TGF-. Alpha.in example 1 of the present invention, b is a graph showing the results of the detection of different concentrations of IFN-. Alpha.2b in 10% human serum (n=3), C is a graph showing the results of the detection of different concentrations of IFN-. Alpha.2b in PBS and 10% human serum (1-3:0.3, 1 and 5 ng/mL), d is a graph showing the relationship between LogΔA652 and LogC of IFN-. Alpha.2b in PBS and 10% human serum;
FIG. 9 is a graph showing the results of ELISA detection of Fe 3O4 @ PEI-anti-body in example 1 of the present invention after storage at 4℃for 0, 10, 20, 30 days.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention provides application of a peroxide nano-enzyme in interferon alpha 2b detection, wherein the peroxide nano-enzyme is a polyethyleneimine modified ferroferric oxide nano-particle.
As a cationic polymer, polyethyleneimine (PEI) has many advantages in Modifying Nanoparticles (MNPs): (1) PEI has good water solubility, physical and chemical stability, and aggregation among MNPs is reduced; (2) The PEI has a lower shielding effect on the catalytic active center of Fe 3O4 MNPs due to the branch structure; (3) The PEI has higher amino content, so that more coupling sites are provided for the antibody; (4) Through surface polymerization, the uniform PEI protective layer can effectively avoid oxidation of Fe 3O4 MNPs.
The average size of the polyethyleneimine modified ferroferric oxide nanoparticles is preferably 100 to 400nm, more preferably 100 to 300nm, and even more preferably 100 to 200nm.
The saturation magnetization value of the polyethyleneimine modified ferroferric oxide nano-particles is preferably 65-70 emu/g, more preferably 66-68 emu/g, and still more preferably 67.11emu/g.
The hydraulic size of the polyethyleneimine modified ferroferric oxide nano-particles is preferably 550-700 nm, more preferably 580-680 nm, even more preferably 590-650 nm, and most preferably 620.97 +/-27.95 nm.
In the present invention, the polyethyleneimine modified ferroferric oxide nanoparticle is preferably prepared according to the following method: and carrying out solvothermal reaction on ferric salt and polyethyleneimine in the presence of a reducing agent and a surfactant to obtain polyethyleneimine modified ferroferric oxide nano particles. Wherein the reducing agent is a reducing agent well known to those skilled in the art, and is not particularly limited, and ethylene glycol is preferred in the present invention; the ratio of the iron salt to the reducing agent is preferably 1g: (30-80) mL, more preferably 1g: (40-60) mL, and more preferably 1g:50mL; the surfactant is preferably one or more of hydrazine, polyvinyl alcohol, sodium acetate, polyvinylpyrrolidone and sodium dodecyl sulfate; the mass ratio of the ferric salt to the surfactant is preferably 1: (2 to 6), more preferably 1: (3 to 5), and more preferably 1:4, a step of; the iron salt is well known to those skilled in the art, and is not particularly limited, and may be an inorganic iron salt or an organic iron salt, preferably one or more of ferric chloride, ferrous sulfate and ferrocene; the weight average molecular weight of the polyethyleneimine is preferably 600 to 25000, more preferably 5000 to 25000, still more preferably 20000 to 25000; the mass ratio of the ferric salt to the polyethyleneimine is 1: (0.5-2); the temperature of the solvothermal reaction is preferably 150-240 ℃, more preferably 180-240 ℃, still more preferably 210-230 ℃, and most preferably 220 ℃; the time of the solvothermal reaction is preferably 1 to 3 hours, more preferably 1.5 to 2.5 hours, still more preferably 2 hours.
In the invention, the polyethyleneimine modified ferroferric oxide nano-particles are prepared according to the following method: mixing ferric salt with a reducing agent, adding a surfactant and polyethyleneimine, heating and stirring, and then carrying out a dissolving solution reaction to obtain polyethyleneimine modified ferroferric oxide nano particles; the temperature of the heating and stirring is preferably 50-70 ℃, more preferably 60 ℃; the heating and stirring time is preferably 10-30 min, more preferably 15-25 min, and still more preferably 20min; the solvothermal reaction is preferably carried out in an autoclave; washing the reaction product with water and ethanol after solvent thermal reaction, separating by magnetic force, and vacuum drying to obtain the polyethyleneimine modified ferroferric oxide nano particles; the temperature of the vacuum drying is preferably 50 to 70 ℃, more preferably 60 ℃.
The invention synthesizes the peroxide nano enzyme which has the advantages of simple preparation method, multiple modification sites, high stability and low cost, can replace the natural enzyme, and is used for sensitive detection of IFN-alpha 2 b.
The invention also provides a detection method of the interferon alpha 2b, which comprises the following steps: dripping a sample to be detected of interferon alpha 2b into a detection plate coated with an interferon alpha 2b antibody for first incubation, adding peroxidase coupled with the interferon alpha 2b antibody for second incubation, adding an auxiliary reagent for third incubation, and detecting absorbance; the peroxide nano-enzyme is a polyethyleneimine modified ferroferric oxide nano-particle.
The sources of all raw materials are not particularly limited, and the raw materials are commercially available; the preparation of the peroxidase is as described above and will not be described in detail here.
Dripping an interferon alpha 2b sample to be detected into an interferon alpha 2b antibody coated detection plate for first incubation; the concentration of the interferon alpha 2b in the interferon alpha 2b sample to be detected is preferably 0.075-25 ng/mL; the interferon alpha 2b antibody coated assay plate is preferably prepared according to the following steps: coating the interferon alpha 2b antibody solution on a detection plate, and after incubation, sealing to obtain the interferon alpha 2b antibody coated detection plate; the concentration of the interferon alpha 2b antibody solution is preferably 10 to 50. Mu.g/mL, more preferably 20 to 30. Mu.g/mL; the temperature of the incubation is preferably 2-6 ℃, more preferably 4 ℃; the incubation time is preferably 8 to 15 hours, more preferably 8 to 12 hours; preferably, the incubation is followed by rinsing and then sealing; the washing is preferably performed by using PBS buffer; the concentration of the PBS buffer is preferably 0.01 to 0.05mol/L, more preferably 0.02 to 0.03mol/L; the pH value of the PBS buffer solution is preferably 6.5-7.5, more preferably 7; the blocking liquid adopted by the blocking is preferably 20% FBS; the closing time is preferably 20-30 hours, more preferably 24 hours; the detection plate may be a well-known plate, and is not particularly limited, but a 96-well plate is preferable in the present invention; the concentration of the interferon alpha 2b in the interferon alpha 2b sample to be detected is preferably 0.075-25 ng/mL; the volume of the interferon alpha 2b sample to be measured is preferably 50-200 mu L, more preferably 80-150 mu L, and still more preferably 100 mu L; the temperature of the first incubation is preferably 36-37 ℃; the first incubation time is preferably 0.5 to 1.5 hours, more preferably 0.8 to 1.2 hours, still more preferably 1 hour.
Washing is preferred after the first incubation, and then peroxide nano enzyme coupled with interferon alpha 2b antibody is added for the second incubation; the washing is preferably performed with PBS buffer; the concentration of the PBS buffer is preferably 0.01 to 0.05mol/L, more preferably 0.02 to 0.03mol/L; the pH value of the PBS buffer solution is preferably 6.5-7.5, more preferably 7; the hydraulical size of the peroxidase conjugated interferon alpha 2b antibody is preferably 650-750 nm, more preferably 670-740 nm, and still more preferably 700.50 +/-30.66 nm; The peroxidase nanoenzyme coupled with the interferon alpha 2b antibody is preferably prepared according to the following method: mixing and reacting the peroxide nano-enzyme with dialdehyde solution to obtain dialdehyde-functionalized peroxide nano-enzyme; mixing dialdehyde functionalized peroxidase with interferon alpha 2b antibody solution, and adding a blocking solution to block to obtain the peroxidase coupled with the interferon alpha 2b antibody; the dialdehyde solution is preferably glutaraldehyde solution; the concentration of the dialdehyde solution is preferably 5% -10% (w/v); the ratio of the peroxide nano-enzyme to the dialdehyde solution is preferably 1mg: (80 to 150) μL, more preferably 1mg: (80-120) mu L, and more preferably 1mg: 100. Mu.L; The mixing reaction time is preferably 3 to 6 hours, more preferably 4 to 5 hours; after the mixing reaction, preferably collecting dialdehyde-functionalized peroxidase, and removing excess dialdehyde by washing; the washing is preferably performed with PBS buffer. Mixing dialdehyde functionalized peroxidase with interferon alpha 2b antibody solution, and adding a blocking solution to block to obtain the peroxidase coupled with the interferon alpha 2b antibody; the concentration of the interferon alpha 2b antibody solution is preferably 0.1-1 mg/mL, more preferably 0.3-0.6 mg/mL, still more preferably 0.4-0.6 mg/mL; The solvent of the interferon alpha 2b antibody solution is preferably PBS buffer solution; the mass ratio of the interferon alpha 2b antibody to the peroxidase nano-enzyme in the interferon alpha 2b antibody solution is preferably (0.1-0.4): 1, more preferably (0.2 to 0.3): 1, a step of; the blocking is carried out by using blocking solution which is well known to the person skilled in the art, preferably by using PBS buffer containing BSA; the mass concentration of BSA in the PBS buffer solution containing BSA is preferably 0.5-2%, more preferably 0.8-1.5%, and even more preferably 1%; the closing time is preferably 0.5 to 2 hours, more preferably 0.8 to 1.5 hours, still more preferably 1 to 1.2 hours; The peroxidase nanoenzyme of the conjugated interferon alpha 2b antibody obtained after the blocking is preferably stored at 4 ℃. The peroxidase nanoenzyme coupled with the interferon alpha 2b antibody is preferably added in the form of a solution thereof; the concentration of the peroxidase-conjugated interferon alpha 2b antibody-containing nano-enzyme solution is preferably 300-1000. Mu.g/mL, more preferably 400-600. Mu.g/mL, still more preferably 500. Mu.g/mL; the solvent of the peroxidase nano-enzyme solution coupled with the interferon alpha 2b antibody is preferably PBS buffer solution; the volume ratio of the peroxidase solution coupled with the interferon alpha 2b antibody to the interferon alpha 2b sample to be detected is preferably 1: (0.8 to 1.2), more preferably 1:1, a step of; The temperature of the second incubation is preferably 36-37 ℃; the time of the second incubation is preferably 10 to 20min, more preferably 15min.
Washing is preferred after the second incubation, and then auxiliary reagent is added for the third incubation; the washing is preferably performed with PBS buffer; the concentration of the PBS buffer is preferably 0.01 to 0.05mol/L, more preferably 0.02 to 0.03mol/L; the pH value of the PBS buffer solution is preferably 6.5-7.5, more preferably 7; the volume ratio of the auxiliary reagent to the interferon alpha 2b sample to be detected is preferably 1: (0.8 to 1.2), more preferably 1:1, a step of; the auxiliary reagent preferably comprises a color developing solution and a buffer solution; the color development liquid preferably comprises color development liquid A and color development liquid B; the color development liquid A is preferably 3,3', 5' -tetramethyl benzidine (TMB) solution; the concentration of the 3,3', 5' -Tetramethylbenzidine (TMB) solution is preferably 5-20 mg/mL, more preferably 8-15 mg/mL, still more preferably 8-12 mg/mL, and most preferably 10mg/mL; the color development liquid B is preferably hydrogen peroxide solution; the mass concentration of the hydrogen peroxide solution is preferably 20-40%, more preferably 25-35%, and even more preferably 30%; the buffer is preferably a NaAC-HAC buffer; the concentration of the NaAC-HAC buffer solution is preferably 100-300 mmol/L, more preferably 150-250 mmol/L, and still more preferably 200mmol/L; the pH of the NaAC-HAC buffer is preferably 3 to 5, more preferably 3 to 4, still more preferably 3.5 to 4, most preferably 3.6; the volume ratio of the color developing solution A to the color developing solution B to the buffer solution is preferably 1: (2-6): (10 to 20), more preferably 1: (3-5): (14 to 16), and more preferably 1:4:15; the temperature of the third incubation is preferably 30-45 ℃, more preferably 35-40 ℃, and most preferably 36-37 ℃; the time for the third incubation is preferably 10 to 20min, more preferably 12 to 18min, still more preferably 14 to 16min, most preferably 15min. According to the invention, polyethyleneimine (PEI) modified Fe 3O4 MNPs(Fe3O4 @PEI MNPs with peroxidase-like activity are synthesized through a one-step method, colorless 3,3', 5' -Tetramethylbenzidine (TMB) is oxidized to blue oxTMB as ELISA signal output for detecting IFN-alpha 2b for the first time as a peroxidase.
Detecting absorbance after the third incubation; in the invention, detecting UV-Vis absorbance in a microplate reader; the wavelength of the detected absorbance is preferably 652nm; the concentration of interferon alpha 2b in the sample to be tested is related to the change in absorbance.
The invention uses the polyethyleneimine modified ferroferric oxide nano-particles as peroxide nano-enzyme to carry out immunoassay on interferon alpha 2b, so that the detection method has the advantages of strong antibody capturing capability, high specificity and sensitivity and low cost.
The invention also provides a detection kit of interferon alpha 2b, which comprises: an interferon alpha 2b antibody coated detection plate, a peroxide nano enzyme coupled with the interferon alpha 2b antibody and an auxiliary reagent;
The peroxide nano enzyme is a polyethyleneimine modified ferroferric oxide nano particle; the peroxidase nanoenzyme coupled with the interferon alpha 2b antibody is preferably mixed in PBS buffer solution; the concentration of the peroxidase conjugated interferon alpha 2b antibody in the PBS buffer solution of the mixed peroxidase conjugated interferon alpha 2b antibody is preferably 300 to 1000. Mu.g/mL, more preferably 400 to 600. Mu.g/mL, still more preferably 500. Mu.g/mL.
Wherein, the peroxidase nano-enzyme coupled with the interferon alpha 2b antibody and the polyethyleneimine modified ferroferric oxide nano-particle are the same as described above, and are not repeated here.
The auxiliary reagent preferably comprises a color developing solution and a buffer solution; the color development liquid comprises color development liquid A and color development liquid B; the color development liquid A is preferably 3,3', 5' -tetramethyl benzidine (TMB) solution; the concentration of the 3,3', 5' -Tetramethylbenzidine (TMB) solution is preferably 5-20 mg/mL, more preferably 8-15 mg/mL, still more preferably 8-12 mg/mL, and most preferably 10mg/mL; the color development liquid B is preferably hydrogen peroxide solution; the mass concentration of the hydrogen peroxide solution is preferably 20-40%, more preferably 25-35%, and even more preferably 30%; the buffer is preferably a NaAC-HAC buffer; the concentration of the NaAC-HAC buffer solution is preferably 100-300 mmol/L, more preferably 150-250 mmol/L, and still more preferably 200mmol/L; the pH of the NaAC-HAC buffer is preferably 3 to 4, more preferably 3.4 to 3.8, still more preferably 3.6.
In order to further illustrate the present invention, the following examples are provided to describe in detail a method and a kit for detecting interferon alpha 2 b.
The reagents used in the examples below are all commercially available.
Example 1
1.1 Preparation of Fe 3O4 @PEI MNPs:
After 1.0g of anhydrous ferric chloride was dissolved in 50mL of ethylene glycol and magnetically stirred to form a yellow transparent solution, 4.0g of anhydrous sodium acetate and 1.0g of polyethyleneimine (PEI having weight average molecular weights of 600, 1800 and 25000, respectively) were added to the solution. The mixture was vigorously stirred at 60℃for 20 minutes, then transferred to an autoclave and reacted at 220℃for 2 hours. The resulting brown-black product was washed three times with water and ethanol, separated with a magnetic rack, and finally dried under vacuum at 60 ℃.
1.2Fe 3O4 @PEI MNPs coupled IFN-. Alpha.2b antibodies
Firstly, fe 3O4 @PEI MNPs are modified by taking glutaraldehyde as a coupling agent: 200. Mu.L of 5% (w/v) glutaraldehyde was added to the pre-prepared Fe 3O4 @PEI MNPs (2 mg), and after continuous stirring for 4 hours, magnetic glutaraldehyde functionalized Fe 3O4 @PEI MNPs were collected. The mixture was washed 3 times with PBS to remove excess glutaraldehyde. Then 1mL (0.4 mg/mL) of IFN-. Alpha.2b antibody PBS buffer was added. Thereafter, the antibody-bound Fe 3O4 @PEI MNPs were blocked with 1% BSA/PBS (2 mL) for 1h and stored at 4 ℃.
1.3 Steady state kinetic analysis of catalysis of Fe 3O4 @PEI MNPs
Kinetic analysis used the following system: 30. Mu.g of Fe 3O4 @PEI MNPs, 832. Mu.M TMB (solvent is DMSO: absolute ethanol volume ratio 1:9) and different concentrations of H 2O2 (0.02-2M) or fixed concentrations of H 2O2 (1.25M) and different concentrations of TMB (41.6-1164.8. Mu.M) were performed in 500. Mu.L NaAC-HAC buffer with pH 3.6. All reaction kinetics were monitored at 652nm using an ultraviolet-visible spectrophotometer. Kinetic parameters were calculated according to Michaelis-Menten equation: v=v max[S]/(Km + [ S ]), where V is the reaction rate; [ S ] is the substrate concentration; v max is the maximum reaction rate and K m is the Michaelis constant. The catalytic constants (k cat)：kcat＝Vmax/[ E ] ([ E ] is the concentration of each enzyme) were calculated, and the catalytic efficiency of Fe 3O4 @ PEI MNPs was calculated as k cat/Km and compared with that of the natural peroxidase HRP.
1.4 Determination of catalytic Activity of Fe 3O4 @PEI MNPs nanoenzyme
In order to better investigate the peroxidase activity before and after antibody modification, catalytic activity comparison studies were performed with a total reaction volume of 500. Mu.L containing Fe 3O4 @PEI MNPs or Fe 3O4 @PEI-anti bodies (30. Mu.g each), respectively. The peroxidase activity of Fe 3O4 @PEI MNPs was measured in different reaction environments such as NaAC-HAC buffer (pH=3.6) containing H 2O2 (1.25M) and TMB (832. Mu.M) at a fixed concentration, at a temperature of 37 ℃ (15 to 55 ℃), pH (3.0 to 8.0), concentration of Fe 3O4 @PEI MNPs (20 to 120. Mu.g/mL) and concentration of H 2O2 (0.02 to 2M).
1.5 ELISA immunoassays for IFN-. Alpha.2b
First, 100. Mu.L IFN-. Alpha.2b antibody (20. Mu.g/mL) was coated on a 96-well plate and incubated overnight at 4 ℃. After 3 washes with 0.02M PBS (pH 7.0), the cells were blocked with 20% FBS for 24h. Then, 100. Mu.L of IFN-. Alpha.2b at various concentrations (0.075-25 ng/mL) was dropped into the wells and incubated at 37℃for 1h. After washing again, 100. Mu.L (500. Mu.g/mL) of the prepared Fe 3O4 @PEI-anti-body was dropped into the well and incubated at 37℃for 15min. After washing, 75. Mu.L of NaAC-HAC buffer (200 mM, pH 3.6), 5. Mu.L of TMB (10 mg/mL) and 20. Mu.L of 30% H 2O2 were added to the wells in this order and incubated at 37℃for 15min. Finally, the 96-well plate is placed in an enzyme-labeled instrument, the UV-Vis absorbance of the sample at 652nm is measured, and the concentration of IFN-alpha 2b is related to the change of absorbance.
1.6 Selective investigation and detection of IFN-alpha 2b in human serum
The interference protein (1 mug/mL) with 100 times of IFN-alpha 2b concentration is added into the detection system: BSA, transforming growth factor-alpha (TGF-alpha) or interleukin-2, 4,6 (IL-2, IL-4, IL-6), the selectivity of the assay was further examined. In actual sample assays, different concentrations of IFN-. Alpha.2b were diluted in 10% human serum and compared to PBS.
Results and analysis
Synthesis and characterization of Fe 3O4 @PEIPMNPs nanomaterials the morphology of Fe 3O4 @PEIPMNPs (PEI weight average molecular weight 25000) was characterized using Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM). As shown in fig. 1a, the shape of the Fe 3O4 @peimnps is spheroidal, and the magnified TEM image shows that its surface has a transparent polymer shell coating (fig. 1 b). The corresponding SEM image (fig. 1 c) shows that Fe 3O4 @ PEI MNPs are surface smooth, highly dispersed spherical nanoparticles with an average size of about 200nm (fig. 1 d). The formation, crystal structure and phase purity of Fe 3O4 @PEI MNPs were verified by X-ray diffraction (XRD) analysis. Fe 3O4 @PEIPIPs and Fe 3O4 MNPs (FIG. 1 e) have similar X-ray diffraction (XRD) spectra, diffraction peaks appear at 30.1 °, 35.4 °, 43.1 °, 53.4 °, 57.0 ° and 62.6 °, belonging to (220), (311), (400), (422), (511) and (440) crystal planes respectively, Is consistent with the cubic structure of Fe 3O4 MNPs (JCPLS No. 75-1609). Fourier transform infrared spectra of Fe 3O4 @ PEI MNPs and Fe 3O4 MNPs were characterized (fig. 1 f), with an adsorption peak at 580cm -1 being Fe-O vibration and a broad strong peak at 3434cm -1 being OH/-NH 2 tensile vibration. In addition, vibration modes at 1082cm -1 and 1633cm -1 are associated with C-N stretching and N-H bending, respectively. These results confirm the successful modification of PEI onto Fe 3O4 MNPs. Furthermore, the Fe 3O4 @PEI MNPs were different in color compared to the black Fe 3O4 MNPs, exhibiting a dark brown color (FIG. 1 g). The saturation magnetization values of Fe 3O4 MNPs and Fe 3O4 @PEI MNPs were 76.13 and 67.11emu/g, respectively (FIG. 1 i). The saturation magnetization of Fe 3O4 @PEI MNPs decreased, indicating that non-magnetic PEI was doped into Fe 3O4 MNPs. it is notable that the Fe 3O4 @PEI MNPs realize rapid magnetic separation within 20s under the action of an external magnetic field (figure 1 h), and can be rapidly dispersed when the external magnet is taken out. Therefore, the Fe 3O4 @PEI MNPs have better superparamagnetism and dispersibility.
Different weight average molecular weight PEI (MW: 600, 1800 and 25000) protected Fe 3O4 nanoparticles, namely Fe 3O4 @PEI MNPs, were synthesized and the detection sensitivity of three nanoparticles to detect different concentrations of IFN-. Alpha.2b (0, 10, 50, 100, 200, 400, 800 ng/mL) was examined, respectively. Fe 3O4 @PEIPIPMNPs (TMB final concentration of 1.04mM, H 2O2 final concentration of 0.1M) catalyze colorless TMB in the presence of H 2O2 to produce blue oxidized TMB (oxTMB) having an absorption peak with maximum absorption at 652 nm. As shown in FIG. 2, the absorption value (OD 652) of oxTMB products catalyzed by three Fe 3O4 @PEIPIPPs gradually increases with increasing concentration of IFN-alpha 2b, wherein the variation of the OD 652 of Fe 3O4 nano particles prepared by taking PEI with the weight average molecular weight of 25000 as a protective agent is largest, which indicates that the catalytic activity of the peroxidic oxidase is strongest, and the Fe 3O4 @PEIPIPPs synthesized by PEI with the molecular weight of 25000 is selected for subsequent experiments.
IFN-alpha 2b antibody modification of Fe 3O4 @PEIPIPs
In order to quantify IFN- α2b protein, an IFN- α2b specific antibody needs to be efficiently bound to the surface of Fe 3O4 @PEI MNPs. Zeta potential and Dynamic Light Scattering (DLS) measurements were first used to verify the effect of modification of the Fe 3O4 @pei MNPs antibodies. In FIG. 3a, there is a significant difference in Zeta potential between Fe 3O4 MNPs、Fe3O4 @PEI MNPs and Fe 3O4 @PEI-anti MNPs. The potentials of Fe 3O4 MNP and Fe 3O4 @PEI MNPs were 6.30.+ -. 0.46 and 15.03.+ -. 0.45mV, respectively. Fe 3O4 @PEIPs have a relatively high Zeta potential due to the PEI generating ionized NH 3+ groups in water. After coupling with the antibody, the zeta potential of Fe 3O4 @PEI MNPs was significantly reduced. This is because Fe 3O4 @PEIPs can be covalently bound to the amino groups of IFN-. Alpha.2b antibodies by cross-linking with glutaraldehyde. As shown in FIG. 3b, the hydraulic size of Fe 3O4 @PEI MNPs measured by DLS was 620.97.+ -. 27.95nm. When it was coupled to an antibody, the size of Fe 3O4 @ PEI-anti-body increased to 700.50.+ -. 30.66nm. As shown in FIG. 3c, fe 3O4 @PEI MNPs have no significant absorption peak, whereas Fe 3O4 @PEI-anti body has an absorption peak at around 280nm from IFN-. Alpha.2b antibodies. the above results indicate that IFN-. Alpha.2b antibodies were successfully conjugated to the surface of Fe 3O4 @PEI MNPs.
Determination of the peroxidase Activity of Fe 3O4 @PEI MNPs
The peroxidase activity of Fe 3O4 @PEI MNPs was examined using colorless TMB as chromogenic substrate. In the presence of H 2O2, fe 3O4 @PEIPIPs catalyze TMB to generate blue oxTMB, and the corresponding maximum peak position of ultraviolet absorption is 652nm. The TMB color change indicates that Fe 3O4 @PEI MNPs have peroxidase activity. FIG. 4a is a graph of the UV-visible absorption spectrum of TMB solution under different reaction conditions, wherein 1 is Fe 3O4 @PEI MNPs,2 is TMB,3 is Fe 3O4 @PEI MNPs+TMB,4 is TMB+H 2O2, 5 is Fe 3O4@PEI MNPs+TMB+H2O2, and the inset shows the different tubes indicating the color change. In the presence of H 2O2, the TMB solution turned blue under the catalysis of Fe 3O4 @PEI MNPs, whereas in the absence of Fe 3O4 @PEI MNPs and H 2O2, the absorption value and color intensity of the TMB solution did not change significantly. As shown in FIG. 4b, the color development reaction exhibited a time dependence. After modification with IFN-alpha 2b antibodies, the catalytic activity of Fe 3O4 @PEIPMNPs was reduced to some extent, which also indicated that IFN-alpha 2b antibodies were coated on the nanoparticles. Considering the time dependence of the color development system, when the trend of the curve is gentle, 15min is selected as the optimal reaction time.
The color reaction is closely related to the nano-enzyme concentration, pH, temperature and H 2O2 concentration. Thus, the present invention conducted the following studies to determine the effect of the above-mentioned key factors on the catalytic process of Fe 3O4 @PEI MNPs to obtain the optimal reaction conditions. As shown in FIG. 5a, as the concentration of Fe 3O4 @ PEI MNPs in the solution increases from 20 μg/mL to 120 μg/mL, the UV-Vis absorbance signal increases, indicating that more TMB is oxidized. The result shows that the reaction rate of Fe 3O4 @PEI MNPs nanoenzyme has a certain correlation with the catalyst dosage. The tests were performed at pH 3.0, 3.6, 4.0, 4.6, 5.0, 5.6, 6.0, 7.0 and 8.0. The results in FIG. 5b show that 3.6 is the optimal pH for the catalytic reaction. As can be seen from FIG. 5c, in the temperature range of 15-55℃the UV-Vis signal increases with increasing temperature, reaches a maximum at 45℃and then decreases. However, considering that the antibody is denatured and inactivated at this temperature, 37℃was selected as the optimal detection temperature for ELISA. As shown in FIG. 5d, when the H 2O2 concentration was below 1.25M, the catalytic activity of Fe 3O4 @PEI MNPs increased first and then a decreasing trend was seen, indicating that high concentrations of H 2O2 inhibited the peroxidase activity of Fe 3O4 @PEI MNPs. Thus, H 2O2 at a concentration of 1.25M was selected for subsequent experiments.
Determination of Fe 3O4 @PEI MNPs catalytic kinetic parameters
The present invention calculates the slope of initial absorbance over time at a fixed catalyst concentration, resulting in steady state reaction rates at different substrate concentrations, and plots a typical Michaelis-Menten curve (FIGS. 6a and b). After fitting the data, a linehaver-Burk plot was established and steady state kinetic parameters were determined (FIGS. 6c and d). as shown in Table 1, the K m value of Fe 3O4 @PEIPIPPs with H 2O2 as substrate was 180mM, which is significantly higher than HRP (3.70 mM) (Nat Nanotechnol 2007,2,577). This result shows that higher concentrations of H 2O2 are required to achieve maximum reaction rates of Fe 3O4 @PEI MNPs. The K m value of Fe 3O4 @PEI MNPs using TMB as a substrate is 0.28mM, which is lower than HRP (0.434 mM) (Nat Nanotechnol 2007,2,577), which indicates that the affinity of Fe 3O4 @PEI MNPs to TMB is higher than HRP. In addition, the invention also compares the catalytic performance of Fe 3O4 @PEI MNPs with that of natural HRP enzyme. calculated by the particle number of Fe 3O4 @PEI MNPs, the k cat/Km value of Fe 3O4 @PEI MNPs to TMB is 1.87 multiplied by 108M -1s-1, The k cat/Km value (9.22×106M -1s-1) above HRP (Nat Nanotechnol2007,2,577) indicates that Fe 3O4 @ PEI MNPs are suitable for ELISA detection.
Table 1 Fe 3O4 @ PEI MNPs vs. HRP kinetic parameters
Fe 3O4 @PEI MNPs combined immune method for detecting IFN-alpha 2b
In view of the peroxidase catalytic properties of Fe 3O4 @PEI MNPs, the present invention uses Fe 3O4 @PEI MNPs in ELISA assays for IFN-. Alpha.2b. FIG. 7a shows that as IFN-. Alpha.2b concentration increases, the absorbance signal increases sharply and then reaches plateau. In fig. 7b, the absorbance change is linearly related to the IFN- α2b concentration in the range of 0.075 to 25ng/mL (R 2 =0.994), with a corresponding detection limit of 0.037ng/mL (blank+3sd, sd: standard deviation). Regression equation is Log ΔA=0.259×log C-0.454 (ΔA=A-A 0、A、A0 is the UV-Vis absorbance at 652nm in the presence or absence of IFN-. Alpha.2b, respectively; log C represents the logarithm of IFN-. Alpha.2b concentration). Thus, the method of the invention has good sensitivity for IFN-alpha 2b detection.
Selectivity investigation and detection of IFN-alpha 2b in serum samples
Selectivity is an important indicator for evaluating the performance of a biosensor. The detection of IFN-. Alpha.2b selectivity based on Fe 3O4 @PEIMNPs was studied by comparing IFN-. Alpha.2b with the UV-visible signal response of interfering proteins such as BSA, IL-2, IL-4, IL-6 and TGF-. Alpha.at 100-fold concentrations. As shown in fig. 8a, IFN- α2b detected a significantly higher signal at a concentration of 10ng/mL than other control protein samples at a concentration of 1 μg/mL (< 0.001) P. In conclusion, the present experiment has higher selectivity for IFN-. Alpha.2b. To investigate the practical application of this strategy in IFN- α2b detection in real samples, concentration determinations were made by adding different concentrations of IFN- α2b (0.075, 0.3, 1, 5, and 25 ng/mL) to 10% human serum. As shown in FIG. 8b (inset: log ΔA652 calibration curve versus Log C for different concentrations of IFN-. Alpha.2b) (IFN-. Alpha.2b concentration: 0.075-25 ng/mL), the variance of UV-Vis absorbance at 652nm increases with increasing IFN-. Alpha.2b concentration the change in UV-Vis absorbance values for IFN-. Alpha.2b at different concentrations was then tested with 10% human serum, and experimental results indicated that there was no significant difference in the detection of IFN-. Alpha.2b under PBS and complex environmental conditions, indicating that the detection of IFN-. Alpha.2b was not substantially disturbed by serum (FIG. 8C). The results of FIG. 8d indicated that Fe 3O4 @ PEI MNPs were suitable for detection of IFN-. Alpha.2b in actual samples.
In addition, IFN-. Alpha.2b was studied in 10% human serum at concentrations of 1, 5 and 25ng/mL, respectively, with recovery ranging from 99.90% to 111.86% (Table 2). These results indicate that the IFN-. Alpha.2b detection strategy provided by the present invention is suitable for analysis of complex biological matrices. Stability is an important parameter in evaluating the performance of chemical immunosensors. As shown in FIG. 9, the prepared Fe 3O4 @PEI MNPs are stable within 30 days, and the relative activity is between 98.69% and 115.24%. The results show that the method is suitable for the determination of IFN-alpha 2b in serum samples, and has potential clinical application value in the pharmacokinetics research of IFN-alpha 2b in antiviral/cancer treatment.
TABLE 2 dilution recovery Experimental results of IFN-. Alpha.2b in human serum
aSD:the standard deviation of three successive measurements.bRecovery(％)＝(Measured value/Expected value)*100.cCV:Coefficient of variation＝SD/Mean measured value.
In conclusion, the invention synthesizes the Fe 3O4 @PEI MNPs with peroxidase activity by adopting a simple and low-cost method. Then, a novel immunoassay for detecting IFN alpha-2 b is provided on the basis, and the synthesized Fe 3O4 @PEI MNPs are used for replacing HRP in the traditional ELISA method. Under optimized conditions, this strategy shows good analytical performance. Fe 3O4 @PEIPIPMNPs mediated IFN- α2b sensing system has been successfully applied to the detection of IFN- α2b in human serum. And systematically explored and demonstrated that the sensing strategy has high selectivity, high sensitivity, high stability and good universality. Therefore, the immunoassay method based on Fe 3O4 @PEI MNPs has great clinical application potential in detecting other protein biomarkers, so that the diagnosis/prognosis or treatment process of various diseases can be remarkably promoted.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (8)
1. A method for detecting interferon alpha 2b, comprising:
Dripping a sample to be detected of interferon alpha 2b into a detection plate coated with an interferon alpha 2b antibody for first incubation, adding peroxidase coupled with the interferon alpha 2b antibody for second incubation, adding an auxiliary reagent for third incubation, and detecting absorbance; the auxiliary reagent comprises a color developing solution and a buffer solution; the color development liquid comprises color development liquid A and color development liquid B; the color development liquid A is 3,3', 5' -tetramethyl benzidine solution; the color development liquid B is hydrogen peroxide solution;
the peroxide nano-enzyme is a polyethyleneimine modified ferroferric oxide nano-particle.
2. The method of claim 1, wherein the polyethyleneimine modified ferroferric oxide nanoparticle is prepared according to the following method:
And carrying out solvothermal reaction on ferric salt and polyethyleneimine in the presence of a reducing agent and a surfactant to obtain polyethyleneimine modified ferroferric oxide nano particles.
3. The method according to claim 2, wherein the reducing agent is ethylene glycol; the surfactant is one or more of hydrazine, polyvinyl alcohol, sodium acetate, polyvinylpyrrolidone and sodium dodecyl sulfate; the ferric salt is selected from one or more of ferric chloride, ferrous sulfate and ferrocene; the mass ratio of the ferric salt to the polyethyleneimine is 1: 0.5-2; the mass ratio of the ferric salt to the surfactant is 1: 2-6; the temperature of the solvothermal reaction is 150-240 ℃; and the solvothermal reaction time is 1-3 h.
4. The method of claim 1, wherein the peroxidase conjugated interferon alpha 2b antibody is prepared by:
Mixing and reacting the peroxide nano-enzyme with dialdehyde solution to obtain dialdehyde-functionalized peroxide nano-enzyme;
And mixing the dialdehyde functionalized peroxidase with the interferon alpha 2b antibody solution, and then adding a blocking solution to block to obtain the peroxidase coupled with the interferon alpha 2b antibody.
5. The method according to claim 4, wherein the dialdehyde solution is glutaraldehyde solution; the concentration of the dialdehyde solution is 5% -10% w/v; the ratio of the peroxide nano enzyme to the dialdehyde solution is 1 mg: 80-150 mu L; the mixing reaction time is 3-6 hours.
6. The detection method according to claim 4, wherein the mass ratio of the interferon alpha 2b antibody to the peroxidase in the interferon alpha 2b antibody solution is 0.1-0.4: 1.
7. The method according to claim 1, wherein the temperature of the first incubation is 36 ℃ to 37 ℃; the first incubation time is 0.5-1.5 h;
The temperature of the second incubation is 36-37 ℃; the second incubation time is 10-20 min;
the temperature of the third incubation is 30-45 ℃; the third incubation time is 10-20 min;
The wavelength of the detected absorbance is 652 nm.
8. A kit for detecting interferon alpha 2b, comprising: an interferon alpha 2b antibody coated detection plate, a peroxide nano enzyme coupled with the interferon alpha 2b antibody and an auxiliary reagent;
the peroxide nano enzyme is a polyethyleneimine modified ferroferric oxide nano particle;
the auxiliary reagent comprises a color developing solution and a buffer solution; the color development liquid comprises color development liquid A and color development liquid B; the color development liquid A is 3,3', 5' -tetramethyl benzidine solution; the color development liquid B is hydrogen peroxide solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310133265.8A CN116068163B (en) | 2023-02-17 | 2023-02-17 | Method and kit for detecting interferon alpha 2b |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310133265.8A CN116068163B (en) | 2023-02-17 | 2023-02-17 | Method and kit for detecting interferon alpha 2b |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116068163A CN116068163A (en) | 2023-05-05 |
| CN116068163B true CN116068163B (en) | 2024-06-25 |
Family
ID=86169698
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310133265.8A Active CN116068163B (en) | 2023-02-17 | 2023-02-17 | Method and kit for detecting interferon alpha 2b |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116068163B (en) |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5262296A (en) * | 1988-03-30 | 1993-11-16 | Toray Industries, Inc. | Freeze-dried composition containing enzyme-labeled anti-human interferon-β antibody and enzyme immunoassay kit containing the composition |
| RU2129275C1 (en) * | 1995-07-06 | 1999-04-20 | Научно-исследовательский институт детских инфекций | Method of determining interferon in infant blood |
| CN103808926B (en) * | 2014-01-14 | 2016-08-17 | 中国科学院生物物理研究所 | Nanometer analogue enztme immunochromatography detection method |
| CN108982834B (en) * | 2018-04-28 | 2021-04-23 | 公安部物证鉴定中心 | Method for detecting biological molecules by using nano enzyme immune sandwich novel technology |
-
2023
- 2023-02-17 CN CN202310133265.8A patent/CN116068163B/en active Active
Non-Patent Citations (1)
| Title |
|---|
| Facile synthesis of Fe3O4@polyethylenimine with peroxidase-like activity for highly sensitive detection of interferon α-2b;Xu Sun et al.;《SCIENCE CHINA Materials》;20230907;第第66卷卷(第第10期期);第4121–4130页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116068163A (en) | 2023-05-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Luo et al. | Fluorescence and magnetic nanocomposite Fe3O4@ SiO2@ Au MNPs as peroxidase mimetics for glucose detection | |
| Li et al. | Immobilization of glucose oxidase onto gold nanoparticles with enhanced thermostability | |
| Chen et al. | A novel bienzyme glucose biosensor based on three-layer Au–Fe3O4@ SiO2 magnetic nanocomposite | |
| Kalyani et al. | Bio-nanocomposite based highly sensitive and label-free electrochemical immunosensor for endometriosis diagnostics application | |
| Xu et al. | Simultaneous electrochemical detection of multiple tumor markers using metal ions tagged immunocolloidal gold | |
| Chang et al. | Pt NPs and DNAzyme functionalized polymer nanospheres as triple signal amplification strategy for highly sensitive electrochemical immunosensor of tumour marker | |
| Ahmadi et al. | An electrochemical immunosensor for digoxin using core–shell gold coated magnetic nanoparticles as labels | |
| Omidfar et al. | A high-sensitivity electrochemical immunosensor based on mobile crystalline material-41–polyvinyl alcohol nanocomposite and colloidal gold nanoparticles | |
| Wang et al. | N-(aminobutyl)-N-(ethylisoluminol) functionalized Fe-based metal-organic frameworks with intrinsic mimic peroxidase activity for sensitive electrochemiluminescence mucin1 determination | |
| Yang et al. | Photoelectrochemical bilirubin biosensor based on Fe3O4/hydroxyapatite/molecularly imprinted polypyrrole nanoparticles | |
| Pei et al. | A sandwich-type electrochemical immunosensor based on RhPt NDs/NH2-GS and Au NPs/PPy NS for quantitative detection hepatitis B surface antigen | |
| Cai et al. | Determination of sulfamethoxazole in foods based on CeO2/chitosan nanocomposite-modified electrodes | |
| Zhu et al. | Amperometric immunosensor for simultaneous detection of three analytes in one interface using dual functionalized graphene sheets integrated with redox-probes as tracer matrixes | |
| Zhang et al. | Electrochemical immunosensor for interferon-γ based on disposable ITO detector and HRP-antibody-conjugated nano gold as signal tag | |
| Khalilzadeh et al. | Reduced graphene oxide decorated with gold nanoparticle as signal amplification element on ultra-sensitive electrochemiluminescence determination of caspase-3 activity and apoptosis using peptide based biosensor | |
| Zhang et al. | Ultrasensitive electrochemical microcystin-LR immunosensor using gold nanoparticle functional polypyrrole microsphere catalyzed silver deposition for signal amplification | |
| Yang et al. | Hollow platinum decorated Fe3O4 nanoparticles as peroxidase mimetic couple with glucose oxidase for pseudobienzyme electrochemical immunosensor | |
| Cheng et al. | Colorimetric and electrochemical (dual) thrombin assay based on the use of a platinum nanoparticle modified metal-organic framework (type Fe-MIL-88) acting as a peroxidase mimic | |
| Zhao et al. | Ionic liquid functionalized graphene based immunosensor for sensitive detection of carbohydrate antigen 15-3 integrated with Cd2+-functionalized nanoporous TiO2 as labels | |
| Yang et al. | Peroxidase-like activity of amino-functionalized magnetic nanoparticles and their applications in immunoassay | |
| CN108344783B (en) | Electrochemical cell sensor and preparation method and application thereof | |
| Wang et al. | Electrochemiluminescence immunosensor based on multifunctional luminol-capped AuNPs@ Fe3O4 nanocomposite for the detection of mucin-1 | |
| Zheng et al. | Multifunctionalized ZIFs nanoprobe-initiated tandem reaction for signal amplified electrochemical immunoassay of carbohydrate antigen 24-2 | |
| Wang et al. | Exonuclease III-assisted triple-amplified electrochemical aptasensor based on PtPd NPs/PEI-rGO for deoxynivalenol detection | |
| Lai et al. | In situ deposition of Prussian blue on mesoporous carbon nanosphere for sensitive electrochemical immunoassay |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant |